Human checkpoint kinase

ABSTRACT

The invention provides a human checkpoint kinase (hChk1) and polynucleotides which identify and encode hChk1. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating or preventing disorders associated with expression of hChk1.

[0001] This application is a continuation application of U.S. Ser. No.09/113,785 filed Jul. 10, 1998, all of which application is herebyincorporated by reference herein.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof a human checkpoint kinase and to the use of these sequences in thediagnosis, treatment, and prevention of cancer and immune disorders.

BACKGROUND OF THE INVENTION

[0003] Kinases regulate many different processes such as cellproliferation, differentiation, and cell signaling by adding phosphategroups to proteins. Reversible protein phosphorylation is the mainstrategy for controlling activities of eukaryotic cells. It is estimatedthat more than 1000 of the 10,000 proteins active in a typical mammaliancell are phosphorylated. The high energy phosphate which drives thisactivation is generally transferred from adenosine triphosphatemolecules (ATP) to a particular protein by protein kinases and removedfrom that protein by protein phosphatases. Phosphorylation occurs inresponse to extracellular signals, cell cycle checkpoints, andenvironmental or nutritional stresses. Protein kinases are roughlydivided into two groups; those that phosphorylate tyrosine residues(protein tyrosine kinases, PTK) and those that phosphorylate serine orthreonine residues (serine/threonine kinases, STK). A few proteinkinases have dual specificity for serine/threonine and tyrosineresidues.

[0004] Almost all kinases contain a similar 250-300 amino acid catalyticdomain containing specific residues and sequence motifs characteristicof the kinase family. (Hardie, G. and Hanks, S. (1995) The ProteinKinase Facts Books, Vol I:7-20 Academic Press, San Diego, Calif.) Inparticular, two protein kinase signature sequences have been identifiedin the kinase domain, the first containing an active site lysine residueinvolved in ATP binding, and the second containing an aspartate residueimportant for catalytic activity. If a protein analyzed includes the twoprotein kinase signatures, the probability of that protein being aprotein kinase is close to 100% (MOTIFS search program, GeneticsComputer Group, Madison, Wis.)

[0005] In the process of cell division, the order and timing of cellcycle transitions is under control of cell cycle checkpoints, regulatorypathways which ensure that critical events such as DNA replication andchromosome segregation are carried out with precision. If DNA isdamaged, e.g. by radiation, a checkpoint pathway is activated thatarrests the cell cycle to provide time for repair. If the damage isextensive, apoptosis is induced. In the absence of such checkpoints, thedamaged DNA is inherited by aberrant cells which may cause proliferativedisorders such as cancer. Protein kinases play an important role in thisprocess. For example, a specific kinase, checkpoint kinase 1 (Chk1), hasbeen identified in yeast and mammals and is activated by DNA damage inyeast. Activation of Chk1 leads to the arrest of the cell at the G2/Mtransition. (Sanchez, Y. et al. (1997) Science 277:1497-1501.)Specifically, Chk1 phosphorylates the cell division cycle phosphataseCDC25, inhibiting its normal function which is to dephosphorylate andactivate the cyclin-dependent kinase Cdc2. Cdc2 activation controls theentry of cells into mitosis. (Peng, C-Y et al. (1997) Science277:1501-1505.) Thus, activation of Chk1 prevents the damaged cell fromentering mitosis.

[0006] The regulation of cell cycle kinases, and in particularcheckpoint kinase, has important implications for the control ofproliferative diseases such as cancer. For example, in response to DNAdamage in mammalian cells, the tumor suppressor p53 acts in a checkpointpathway for cell cycle control by inducing the transcription of acyclin-dependent kinase inhibitor resulting in G1/S arrest. (Sanchez etal., supra) p53-deficient cells with damaged DNA are unable to arrest inG1/S, a condition which can lead to cancer. It has been estimated thatp53 may be non-functional in at least 60% of human cancers. (Greenblatt,M. S. et al. (1994) Cancer Research 54:4855-4878.) A similar deficiencyin a checkpoint kinase, such as Chk1, may also contribute to cancer byfailure to arrest cells with damaged DNA at other checkpoints such asG2/M.

[0007] Furthermore, it has been reported that in p53-deficient tumorcells, agents known to override G2/M arrest, specificallymethylxanthines such as caffeine, induce a selective sensitization ofthese cells to agents which damage DNA (radiation or DNA cross-linking,chemotherapeutic agents). (Powell, S. N. et al. (1995) Cancer Research55:1643-1648.) Thus, agents which can override G2/M arrest may be usefulradiosensitizing or chemosensitizing agents for cancer treatment. SinceChk1 has been shown to induce G2/M arrest, Chk1 inhibitors may be usefulin this regard.

[0008] The discovery of a new human checkpoint kinase and thepolynucleotides encoding it satisfies a need in the art by providing newcompositions which are useful in the diagnosis, treatment, andprevention of cancer and immune disorders.

SUMMARY OF THE INVENTION

[0009] The invention is based on the discovery of a new human checkpointkinase (hChk1), the polynucleotides encoding hChk1, and the use of thesecompositions for the diagnosis, treatment, or prevention of cancer andimmune disorders.

[0010] The invention features a substantially purified polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQID NO:1.

[0011] The invention further provides a substantially purified varianthaving at least 90% amino acid sequence identity to the amino acidsequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention alsoprovides an isolated and purified polynucleotide encoding thepolypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQID NO:1. The invention also includes an isolated and purifiedpolynucleotide variant having at least 70% polynucleotide sequenceidentity to the polynucleotide encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. Theinvention further provides an isolated and purified polynucleotidecomprising the polynucleotide sequence of SEQ ID NO:16, or a fragment ofSEQ ID NO:16.

[0012] The invention further provides an isolated and purifiedpolynucleotide which hybridizes under stringent conditions to thepolynucleotide encoding the polypeptide comprising the amino acidsequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as anisolated and purified polynucleotide which is complementary to thepolynucleotide encoding the polypeptide comprising the amino acidsequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

[0013] The invention also provides an isolated and purifiedpolynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 ora fragment of SEQ ID NO:2, and an isolated and purified polynucleotidevariant having at least 70% polynucleotide sequence identity to thepolynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 ora fragment of SEQ ID NO:2. The invention also provides an isolated andpurified polynucleotide having a sequence complementary to thepolynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 ora fragment of SEQ ID NO:2.

[0014] The invention further provides an expression vector comprising atleast a fragment of the polynucleotide encoding the polypeptidecomprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. Inanother aspect, the expression vector is contained within a host cell.

[0015] The invention also provides a method for producing a polypeptide,the method comprising the steps of: (a) culturing the host cellcomprising an expression vector containing at least a fragment of apolynucleotide encoding the polypeptide comprising the amino acidsequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 under conditionssuitable for the expression of the polypeptide; and (b) recovering thepolypeptide from the host cell culture.

[0016] The invention also provides a pharmaceutical compositioncomprising a substantially purified polypeptide having the sequence ofSEQ ID NO:1 or a fragment of SEQ ID NO:1 in conjunction with a suitablepharmaceutical carrier.

[0017] The invention further includes a purified antibody which binds toa polypeptide comprising the sequence of SEQ ID NO:1 or a fragment ofSEQ ID NO:1, as well as a purified agonist and a purified antagonist orinhibitor of the polypeptide.

[0018] The invention also provides a method for treating or preventing acancer, the method comprising administering to a subject in need of suchtreatment an effective amount of a pharmaceutical composition comprisinga substantially purified polypeptide having the amino acid sequence ofSEQ ID NO:1 or a fragment of SEQ ID NO:1.

[0019] The invention also provides a method for treating or preventingan immune disorder, the method comprising administering to a subject inneed of such treatment an effective amount of a pharmaceuticalcomposition comprising a substantially purified polypeptide having theamino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

[0020] The invention also provides a method for treating a cancer, themethod comprising administering to a subject in need of such treatmentan effective amount of an antagonist or inhibitor of the polypeptidehaving the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ IDNO:1, either alone or in combination with radiation or chemotherapy.

[0021] The invention also provides a method for detecting apolynucleotide in a sample containing nucleic acids, the methodcomprising the steps of: (a) hybridizing the complement of thepolynucleotide encoding the polypeptide comprising the amino acidsequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 to at least onenucleic acid in the sample, thereby forming a hybridization complex; and(b) detecting the hybridization complex, wherein the presence of thehybridization complex correlates with the presence of a polynucleotidein the sample. In one aspect, this method further comprises amplifyingthe polynucleotide prior to hybridization.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

[0022]FIGS. 1A, 1B, 1C, and 1D show the amino acid sequence (SEQ IDNO:1) and nucleic acid sequence (SEQ ID NO:2) of hChk1. The alignmentwas produced using MacDNASIS PRO software (Hitachi Software Engineering,South San Francisco Calif.).

[0023]FIGS. 2A, 2B, and 2C show the amino acid sequence alignmentsbetween hChk1 (516219; SEQ ID NO:1), and checkpoint kinase, Chk1 fromyeast (GI 311176; SEQ ID NO:3), produced using the multisequencealignment program of LASERGENE software (DNASTAR Inc, Madison Wis.).

[0024]FIG. 3 shows the kinase activity of purified hChk1 in vitroagainst various natural and synthetic substrates. The Y axis representsradioactivity (cpm) incorporated into phosphorylated products, and the Xaxis, various synthetic peptide substrates as follows: (1) mylein basicprotein (MBP), (2) casein, (3) phosviotin, (4) poly (Arg, Pro, Thr)6:3:1, (5) poly (Arg, Ser) 3:1, (6) poly (Arg, Pro, Thr) 1:1:1, (7) MBPfragment, (8) cAMP substrate 1 (RRKASGP), (9) cAMP substrate 2(GRGLSLSR), (10) protein kinase C substrate, (11) calmodulin substrate,(12) Kemptide, (13) cAMP substrate 3 (LRRWSLG).

[0025]FIG. 4 shows the northern analysis for the nucleotide sequence(SEQ ID NO:2) of hChk1 produced electronically using the LIFESEQdatabase (Incyte Genomics, Palo Alto Calif.).

[0026] Table 1 shows the programs/algorithms, descriptions, referencesand threshold parameters used to identify and characterize hChk1.

DESCRIPTION OF THE INVENTION

[0027] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0028] It must be noted that as used herein and in the appended claims,the singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a host cell” includes a plurality of such host cells, and areference to “an antibody” is a reference to one or more antibodies andequivalents thereof known to those skilled in the art, and so forth.

[0029] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are cited for the purpose of describing and disclosingthe cell lines, vectors, and methodologies which are reported in thepublications and which might be used in connection with the invention.Nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

[0030] Definitions

[0031] “hChk1,” as used herein, refers to the amino acid sequences ofsubstantially purified hChk1 obtained from any species, particularly amammalian species, including bovine, ovine, porcine, murine, equine, andpreferably the human species, from any source, whether natural,synthetic, semi-synthetic, or recombinant.

[0032] The term “agonist,” as used herein, refers to a molecule which,when bound to hChk1, increases or prolongs the duration of the effect ofhChk1. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to and modulate the effect of hChk1.

[0033] An “allelic variant,” as this term is used herein, is analternative form of the gene encoding hChk1. Allelic variants may resultfrom at least one mutation in the nucleic acid sequence and may resultin altered mRNAs or in polypeptides whose structure or function may ormay not be altered. Any given natural or recombinant gene may have none,one, or many allelic forms. Common mutational changes which give rise toallelic variants are generally ascribed to natural deletions, additions,or substitutions of nucleotides. Each of these types of changes mayoccur alone, or in combination with the others, one or more times in agiven sequence.

[0034] “Altered” nucleic acid sequences encoding hChk1, as describedherein, include those sequences with deletions, insertions, orsubstitutions of different nucleotides, resulting in a polynucleotidethe same as hChk1 or a polypeptide with at least one functionalcharacteristic of hChk1. Included within this definition arepolymorphisms which may or may not be readily detectable using aparticular oligonucleotide probe of the polynucleotide encoding hChk1,and improper or unexpected hybridization to allelic variants, with alocus other than the normal chromosomal locus for the polynucleotidesequence encoding hChk1. The encoded protein may also be “altered,” andmay contain deletions, insertions, or substitutions of amino acidresidues which produce a silent change and result in a functionallyequivalent hChk1. Deliberate amino acid substitutions may be made on thebasis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues, as longas the biological or immunological activity of hChk1 is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid, positively charged amino acids may include lysine andarginine, and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline; glycine and alanine; asparagine and glutamine; serine andthreonine; and phenylalanine and tyrosine.

[0035] The terms “amino acid” or “amino acid sequence,” as used herein,refer to an oligopeptide, peptide, polypeptide, or protein sequence, ora fragment of any of these, and to naturally occurring or syntheticmolecules. In this context, “fragments,” “immunogenic fragments,” or“antigenic fragments” refer to fragments of hChk1 which are preferablyat least 5 to about 15 amino acids in length, most preferably at least14 amino acids, and which retain some biological activity orimmunological activity of hChk1. Where “amino acid sequence” is recitedherein to refer to an amino acid sequence of a naturally occurringprotein molecule, “amino acid sequence” and like terms are not meant tolimit the amino acid sequence to the complete native amino acid sequenceassociated with the recited protein molecule.

[0036] “Amplification,” as used herein, relates to the production ofadditional copies of a nucleic acid sequence. Amplification is generallycarried out using polymerase chain reaction (PCR) technologies wellknown in the art. (See, e.g., Dieffenbach, C. W. and G. S. Dveksler(1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y., pp.1-5.)

[0037] The term “antagonist,” as it is used herein, refers to a moleculewhich, when bound to hChk1, decreases or prevents the activity or effectof hChk1. Antagonists may include proteins, nucleic acids,carbohydrates, antibodies, organic molecules (whether naturallyoccuring, semi-synthetic or totally synthetic), or any other moleculeswhich bind to and modulate the effect of hChk1.

[0038] As used herein, the term “antibody” refers to intact molecules aswell as to fragments thereof, such as Fab, F(ab′)₂, and Fv fragments,which are capable of binding the epitopic determinant. Antibodies thatbind hChk1 polypeptides can be prepared using intact polypeptides orusing fragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

[0039] The term “antigenic determinant,” as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or a fragment of a protein is usedto immunize a host animal, numerous regions of the protein may inducethe production of antibodies which bind specifically to antigenicdeterminants (given regions or three-dimensional structures on theprotein). An antigenic determinant may compete with the intact antigen(i.e., the immunogen used to elicit the immune response) for binding toan antibody.

[0040] The term “antisense,” as used herein, refers to any compositioncontaining a nucleic acid sequence which is complementary to the “sense”strand of a specific nucleic acid sequence. Antisense molecules may beproduced by any method including synthesis or transcription. Onceintroduced into a cell, the complementary nucleotides combine withnatural sequences produced by the cell to form duplexes and to blockeither transcription or translation. The designation “negative” canrefer to the antisense strand, and the designation “positive” can referto the sense strand.

[0041] As used herein, the term “biologically active,” refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic hChk1, or ofany oligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0042] The terms “complementary” or “complementarity,” as used herein,refer to the natural binding of polynucleotides by base pairing. Forexample, the sequence “5′ A-G-T 3′” binds to the complementary sequence“3′ T-C-A 5′.” Complementarity between two single-stranded molecules maybe “partial,” such that only some of the nucleic acids bind, or it maybe “complete,” such that total complementarity exists between the singlestranded molecules. The degree of complementarity between nucleic acidstrands has significant effects on the efficiency and strength of thehybridization between the nucleic acid strands. This is of particularimportance in amplification reactions, which depend upon binding betweennucleic acids strands, and in the design and use of peptide nucleic acid(PNA) molecules.

[0043] A “composition comprising a given polynucleotide sequence,” or a“composition comprising a given amino acid sequence,” as these terms areused herein, refer broadly to any composition containing the givenpolynucleotide or amino acid sequence. The composition may comprise adry formulation or an aqueous solution. Compositions comprisingpolynucleotide sequences encoding hChk1 or fragments of hChk1 may beemployed as hybridization probes. The probes may be stored infreeze-dried form and may be associated with a stabilizing agent such asa carbohydrate. In hybridizations, the probe may be deployed in anaqueous solution containing salts, e.g., NaCl, detergents, e.g., sodiumdodecyl sulfate (SDS), and other components, e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.

[0044] “Consensus sequence,” as used herein, refers to a nucleic acidsequence which has been resequenced to resolve uncalled bases, extendedusing XL-PCR (Applied Biosystems, Foster City Calif.) in the 5′ and/orthe 3′ direction, and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte Clone using a computerprogram for fragment assembly, such as the GELVIEW Fragment Assemblysystem (GCG, Madison, Wis.). Some sequences have been both extended andassembled to produce the consensus sequence.

[0045] As used herein, the term “correlates with expression of apolynucleotide” indicates that the detection of the presence of nucleicacids, the same or related to a nucleic acid sequence encoding hChk1, byNorthern analysis is indicative of the presence of nucleic acidsencoding hChk1 in a sample, and thereby correlates with expression ofthe transcript from the polynucleotide encoding hChk1.

[0046] A “deletion,” as the term is used herein, refers to a change inthe amino acid or nucleotide sequence that results in the absence of oneor more amino acid residues or nucleotides.

[0047] The term “derivative,” as used herein, refers to the chemicalmodification of a polypeptide sequence, or a polynucleotide sequence.Chemical modifications of a polynucleotide sequence can include, forexample, replacement of hydrogen by an alkyl, acyl, or amino group. Aderivative polynucleotide encodes a polypeptide which retains at leastone biological or immunological function of the natural molecule. Aderivative polypeptide is one modified by glycosylation, pegylation, orany similar process that retains at least one biological orimmunological function of the polypeptide from which it was derived.

[0048] The term “similarity,” as used herein, refers to a degree ofcomplementarity. There may be partial similarity or complete similarity.The word “identity” may substitute for the word “similarity.” Apartially complementary sequence that at least partially inhibits anidentical sequence from hybridizing to a target nucleic acid is referredto as “substantially similar.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or Northern blot, solutionhybridization, and the like) under conditions of reduced stringency. Asubstantially similar sequence or hybridization probe will compete forand inhibit the binding of a completely similar (identical) sequence tothe target sequence under conditions of reduced stringency. This is notto say that conditions of reduced stringency are such that non-specificbinding is permitted, as reduced stringency conditions require that thebinding of two sequences to one another be a specific (i.e., aselective) interaction. The absence of non-specific binding may betested by the use of a second target sequence which lacks even a partialdegree of complementarity (e.g., less than about 30% similarity oridentity). In the absence of non-specific binding, the substantiallysimilar sequence or probe will not hybridize to the secondnon-complementary target sequence.

[0049] The phrases “percent identity” or “% identity” refer to thepercentage of sequence similarity found in a comparison of two or moreamino acid or nucleic acid sequences. Percent identity can be determinedelectronically, e.g., by using the MEGALIGN program (DNASTAR, Inc.,Madison Wis.). The MEGALIGN program can create alignments between two ormore sequences according to different methods, e.g., the clustal method.(See, e.g., Higgins, D. G. and P. M. Sharp (1988) Gene 73:237-244.) Theclustal algorithm groups sequences into clusters by examining thedistances between all pairs. The clusters are aligned pairwise and thenin groups. The percentage similarity between two amino acid sequences,e.g., sequence A and sequence B, is calculated by dividing the length ofsequence A, minus the number of gap residues in sequence A, minus thenumber of gap residues in sequence B, into the sum of the residuematches between sequence A and sequence B, times one hundred. Gaps oflow or of no similarity between the two amino acid sequences are notincluded in determining percentage similarity. Percent identity betweennucleic acid sequences can also be counted or calculated by othermethods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein,J. (1990) Methods Enzymol. 183:626-645.) Identity between sequences canalso be determined by other methods known in the art, e.g., by varyinghybridization conditions.

[0050] “Human artificial chromosomes” (HACs), as described herein, arelinear microchromosomes which may contain DNA sequences of about 6 kb to10 Mb in size, and which contain all of the elements required for stablemitotic chromosome segregation and maintenance. (See, e.g., Harrington,J. J. et al. (1997) Nat Genet. 15:345-355.)

[0051] The term “humanized antibody,” as used herein, refers to antibodymolecules in which the amino acid sequence in the non-antigen bindingregions has been altered so that the antibody more closely resembles ahuman antibody, and still retains its original binding ability.

[0052] “Hybridization,” as the term is used herein, refers to anyprocess by which a strand of nucleic acid binds with a complementarystrand through base pairing.

[0053] As used herein, the term “hybridization complex” refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary bases. A hybridizationcomplex may be formed in solution (e.g., Cot or Rot analysis) or formedbetween one nucleic acid sequence present in solution and anothernucleic acid sequence immobilized on a solid support (e.g., paper,membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

[0054] The words “insertion” or “addition,” as used herein, refer tochanges in an amino acid or nucleotide sequence resulting in theaddition of one or more amino acid residues or nucleotides,respectively, to the sequence found in the naturally occurring molecule.

[0055] “Immune response” can refer to conditions associated withinflammation, trauma, immune disorders, or infectious or geneticdisease, etc. These conditions can be characterized by expression ofvarious factors, e.g., cytokines, chemokines, and other signalingmolecules, which may affect cellular and systemic defense systems.

[0056] The term “microarray,” as used herein, refers to an arrangementof distinct polynucleotides arrayed on a substrate, e.g., paper, nylonor any other type of membrane, filter, chip, glass slide, or any othersuitable solid support.

[0057] The terms “element” or “array element” as used herein in amicroarray context, refer to hybridizable polynucleotides arranged onthe surface of a substrate.

[0058] The term “modulate,” as it appears herein, refers to a change inthe activity of hChk1. For example, modulation may cause an increase ora decrease in protein activity, binding characteristics, or any otherbiological, functional, or immunological properties of hChk1.

[0059] The phrases “nucleic acid” or “nucleic acid sequence,” as usedherein, refer to a nucleotide, oligonucleotide, polynucleotide, or anyfragment thereof. These phrases also refer to DNA or RNA of genomic orsynthetic origin which may be single-stranded or double-stranded and mayrepresent the sense or the antisense strand, to peptide nucleic acid(PNA), or to any DNA-like or RNA-like material. In this context,“fragments” refers to those nucleic acid sequences which, whentranslated, would produce polypeptides retaining some functionalcharacteristic, e.g., antigenicity, or structural domain characteristic,e.g., ATP-binding site, of the full-length polypeptide.

[0060] The terms “operably associated” or “operably linked,” as usedherein, refer to functionally related nucleic acid sequences. A promoteris operably associated or operably linked with a coding sequence if thepromoter controls the translation of the encoded polypeptide. Whileoperably associated or operably linked nucleic acid sequences can becontiguous and in the same reading frame, certain genetic elements,e.g., repressor genes, are not contiguously linked to the sequenceencoding the polypeptide but still bind to operator sequences thatcontrol expression of the polypeptide.

[0061] The term “oligonucleotide,” as used herein, refers to a nucleicacid sequence of at least about 6 nucleotides to 60 nucleotides,preferably about 15 to 30 nucleotides, and most preferably about 20 to25 nucleotides, which can be used in PCR amplification or in ahybridization assay or microarray. As used herein, the term“oligonucleotide” is substantially equivalent to the terms “amplimer,”“primer,” “oligomer,” and “probe,” as these terms are commonly definedin the art.

[0062] “Peptide nucleic acid” (PNA), as used herein, refers to anantisense molecule or anti-gene agent which comprises an oligonucleotideof at least about 5 nucleotides in length linked to a peptide backboneof amino acid residues ending in lysine. The terminal lysine conferssolubility to the composition. PNAs preferentially bind complementarysingle stranded DNA or RNA and stop transcript elongation, and may bepegylated to extend their lifespan in the cell. (See, e.g., Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63.)

[0063] The term “sample,” as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acids encodinghChk1, or fragments thereof, or hChk1 itself, may comprise a bodilyfluid; an extract from a cell, chromosome, organelle, or membraneisolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution orbound to a solid support; a tissue; a tissue print; etc.

[0064] As used herein, the terms “specific binding” or “specificallybinding” refer to that interaction between a protein or peptide and anagonist, an antibody, or an antagonist. The interaction is dependentupon the presence of a particular structure of the protein, e.g., theantigenic determinant or epitope, recognized by the binding molecule.For example, if an antibody is specific for epitope “A,” the presence ofa polypeptide containing the epitope A, or the presence of freeunlabeled A, in a reaction containing free labeled A and the antibodywill reduce the amount of labeled A that binds to the antibody.

[0065] As used herein, the term “stringent conditions” refers toconditions which permit hybridization between polynucleotides and theclaimed polynucleotides. Stringent conditions can be defined by saltconcentration, the concentration of organic solvent, e.g., formamide,temperature, and other conditions well known in the art. In particular,stringency can be increased by reducing the concentration of salt,increasing the concentration of formamide, or raising the hybridizationtemperature.

[0066] The term “substantially purified,” as used herein, refers tonucleic acid or amino acid sequences that are removed from their naturalenvironment and are isolated or separated, and are at least about 60%free, preferably about 75% free, and most preferably about 90% free fromother components with which they are naturally associated.

[0067] A “substitution,” as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0068] “Transformation,” as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. Transformation mayoccur under natural or artificial conditions according to variousmethods well known in the art, and may rely on any known method for theinsertion of foreign nucleic acid sequences into a prokaryotic oreukaryotic host cell. The method for transformation is selected based onthe type of host cell being transformed and may include, but is notlimited to, viral infection, electroporation, heat shock, lipofection,and particle bombardment. The term “transformed” cells includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, as well as transiently transformed cells which express theinserted DNA or RNA for limited periods of time.

[0069] A “variant” of hChk1 polypeptides, as used herein, refers to anamino acid sequence that is altered by one or more amino acid residues.The variant may have “conservative” changes, wherein a substituted aminoacid has similar structural or chemical properties (e.g., replacement ofleucine with isoleucine). More rarely, a variant may have“nonconservative” changes (e.g., replacement of glycine withtryptophan). Analogous minor variations may also include amino aciddeletions or insertions, or both. Guidance in determining which aminoacid residues may be substituted, inserted, or deleted withoutabolishing biological or immunological activity may be found usingcomputer programs well known in the art, for example, LASERGENE™software.

[0070] The term “variant,” when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to hChk1. Thisdefinition may also include, for example, “allelic” (as defined above),“splice,” “species,” or “polymorphic” variants. A splice variant mayhave significant identity to a reference molecule, but will generallyhave a greater or lesser number of polynucleotides due to alternatesplicing of exons during mRNA processing. The corresponding polypeptidemay possess additional functional domains or an absence of domains.Species variants are polynucleotide sequences that vary from one speciesto another. The resulting polypeptides generally will have significantamino acid identity relative to each other. A polymorphic variant is avariation in the polynucleotide sequence of a particular gene betweenindividuals of a given species. Polymorphic variants also may encompass“single nucleotide polymorphisms” (SNPs) in which the polynucleotidesequence varies by one base. The presence of SNPs may be indicative of,for example, a certain population, a disease state, or a propensity fora disease state.

[0071] The Invention

[0072] The invention is based on the discovery of a new human checkpointkinase (hChk1), the polynucleotides encoding hChk1, and the use of thesecompositions for the diagnosis, treatment, or prevention of cancer andimmune disorders.

[0073] Nucleic acids encoding the hChk1 of the present invention werefirst identified in Incyte Clones 516219 from the mononuclear cell cDNAlibrary (MMLR1DT01) and 2044650 from the promonocyte cell cDNA library(THP1T7T01) using a computer search, e.g., BLAST, for amino acidsequence alignments. A consensus sequence, SEQ ID NO:2, was derived fromthe following overlapping and/or extended nucleic acid sequences: IncyteClones 046081H1, 046081F1 and 046081X3 (CORNNOT01), 516219H1, 516219F1and 516219X5 (MMLR1DT01) 2044650H1 (THP1T7T01) and 2731758H1(OVARTUT04); SEQ ID Nos: 4-11, respectively.

[0074] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A,1B, 1C, and 1D. hChk1 is 476 amino acids in length and has potentialphosphorylation sites for cAMP- and cGMP-dependent protein kinase as atresidue T378, for casein kinase II at T14, S88, T127, S214, T255, S296,S307, S317, S326, S333, T348, T382, S415, and S444, for protein kinase Cat T127, S251, T417, and S468, and for tyrosine kinase at Y390 and Y402.hChk1 has two signature sequences for protein kinases located betweenresidues L15 and K38 and residues 1126 and L138. The first sequence islocated in subdomain I of the potential kinase domain and contains thecharacteristic motif, GXGXXGXV, between residues G16 and V21, and theATP-binding residue, K38. The second sequence located in subdomain VIcontains the consensus motif HRDLKXXN with a conservative substitutionof I for L at residue 131, and the important aspartic acid, catalyticactive site residue, D130. As shown in FIGS. 2A, 2B, and 2C, hChk1 haschemical and structural similarity with checkpoint kinase from yeast,Chk1 (GI 311176; SEQ ID NO:3). In particular, hChk1 and Chk1 share 23%identity. The two proteins share the consensus motif GXGXXGXV insubdomain I, the ATP-binding residue at K38, the consensus motifHRDLKXXN in subdomain VI, and the catalytic active site residue, D130.

[0075] The nucleotide sequence encoding hChk1 was expressed in insectcells using a baculovirus vector and hChk1 was isolated and prepared forprotein activity assays. The expression of the protein was assessed bywestern analysis using an antibody prepared against a C-terminal portionof the polypeptide (CGKIKGKLIDIVSSQKVWLPAT; SEQ ID NO:12). FIG. 3 showsthe results of an in vitro protein kinase phosphorylation assay usinghChk1 incubated with various proteins or synthetic peptide substratesand [γ-³³P]ATP as the phosphate donor. Control reactions were carriedout in the absence of hChk1. hChk1 showed the highest phosphorylatingactivity using a calmodulin substrate or the synthetic peptidesubstrate, poly (Arg, Ser) 3:1, but significant activity was also foundwith mylein basic protein (MBP), casein, poly (Arg, Pro, Thr) 6:3:1,protein kinase C (PKC) substrate, and cAMP substrate.

[0076] Electronic northern analysis (FIG. 4) using the LIFESEQ databaseshowed the expression of this sequence in various libraries, at least67% of which are immortalized or cancerous and at least 38% of whichinvolve immune response. Of particular note is the expression of hChk1in cancers of the brain, ovaries, lymph nodes, colon, testis, andleukemia.

[0077] The invention also encompasses hChk1 variants. A preferred hChk1variant is one which has at least about 80%, more preferably at leastabout 90%, and most preferably at least about 95% amino acid sequenceidentity to the hChk1 amino acid sequence, and which contains at leastone functional or structural characteristic of hChk1.

[0078] The invention also encompasses polynucleotides which encodehChk1. In a particular embodiment, the invention encompasses apolynucleotide sequence comprising the sequence of SEQ ID NO:2, whichencodes an hChk1.

[0079] The invention also encompasses a variant of a polynucleotidesequence encoding hChk1. In particular, such a variant polynucleotidesequence will have at least about 70%, more preferably at least about85%, and most preferably at least about 95% polynucleotide sequenceidentity to the polynucleotide sequence encoding hChk1. A particularaspect of the invention encompasses a variant of SEQ ID NO:2 which hasat least about 70%, more preferably at least about 85%, and mostpreferably at least about 95% polynucleotide sequence identity to SEQ IDNO:2. Any one of the polynucleotide variants described above can encodean amino acid sequence which contains at least one functional orstructural characteristic of hChk1.

[0080] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude ofpolynucleotide sequences encoding hChk1, some bearing minimal similarityto the polynucleotide sequences of any known and naturally occurringgene, may be produced. Thus, the invention contemplates each and everypossible variation of polynucleotide sequence that could be made byselecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode as applied to the polynucleotide sequence of naturally occurringhChk1, and all such variations are to be considered as beingspecifically disclosed.

[0081] Although nucleotide sequences which encode hChk1 and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring hChk1 under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding hChk1 or its derivatives possessing a substantially differentcodon usage, e.g., inclusion of non-naturally occurring codons. Codonsmay be selected to increase the rate at which expression of the peptideoccurs in a particular prokaryotic or eukaryotic host in accordance withthe frequency with which particular codons are utilized by the host.Other reasons for substantially altering the nucleotide sequenceencoding hChk1 and its derivatives without altering the encoded aminoacid sequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

[0082] The invention also encompasses production of DNA sequences whichencode hChk1 and hChk1 derivatives, or fragments thereof, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents well known in the art. Moreover, syntheticchemistry may be used to introduce mutations into a sequence encodinghChk1 or any fragment thereof.

[0083] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed polynucleotide sequences,and, in particular, to that shown in SEQ ID NO:2, or a fragment of SEQID NO:2, under various conditions of stringency. (See, e.g., Wahl, G. M.and S. L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R.(1987) Methods Enzymol. 152:507-511.) For example, stringent saltconcentration will ordinarily be less than about 750 mM NaCl and 75 mMtrisodium citrate, preferably less than about 500 mM NaCl and 50 mMtrisodium citrate, and most preferably less than about 250 mM NaCl and25 mM trisodium citrate. Low stringency hybridization can be obtained inthe absence of organic solvent, e.g., formamide, while high stringencyhybridization can be obtained in the presence of at least about 35%formamide, and most preferably at least about 50% formamide. Stringenttemperature conditions will ordinarily include temperatures of at leastabout 30° C., more preferably of at least about 37° C., and mostpreferably of at least about 42° C. Varying additional parameters, suchas hybridization time, the concentration of detergent, e.g., sodiumdodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA,are well known to those skilled in the art. Various levels of stringencyare accomplished by combining these various conditions as needed. In apreferred embodiment, hybridization will occur at 30° C. in 750 mM NaCl,75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment,hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodiumcitrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA(ssDNA). In a most preferred embodiment, hybridization will occur at 42°C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and200 μg/ml ssDNA. Useful variations on these conditions will be readilyapparent to those skilled in the art.

[0084] The washing steps which follow hybridization can also vary instringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude temperature of at least about 25° C., more preferably of atleast about 42° C., and most preferably of at least about 68° C. In apreferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art.

[0085] Methods for DNA sequencing are well known in the art and may beused to practice any of the embodiments of the invention. The methodsmay employ such enzymes as the Klenow fragment of DNA polymerase I,SEQUENASE (US Biochemical Corp., Cleveland, Ohio), Taq polymerase(Applied Biosystems), thermostable T7 polymerase (Amersham Biosciences(APB), Piscataway N.J.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE Amplification System(Invitrogen, San Diego ca). Preferably, sequence preparation isautomated with machines such as the Hamilton Micro Lab 2200 (Hamilton,Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown,Mass.) and the ABI Catalyst 800 (Applied Biosystems). Sequencing is thencarried out using either ABI 373 or 377 DNA Sequencing Systems (AppliedBiosystems) or capillary electrophoresis (Molecular Dynamics). Theresulting sequences are analyzed using a variety of alogorithms whichare well known in the art. (See, e.g., Ausubel, supra, ch. 7.7; andMeyers, R. A. (1995) Molecular Biology and Biotechnology Wiley VCH,Inc., New York, N.Y., pp. 856-853.)

[0086] The nucleic acid sequences encoding hChk1 may be extendedutilizing a partial nucleotide sequence and employing various PCR-basedmethods known in the art to detect upstream sequences, such as promotersand regulatory elements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector. (See, e.g.,Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method,inverse PCR, uses primers that extend in divergent directions to amplifyunknown sequence from a circularized template. The template is derivedfrom restriction fragments comprising a known genomic locus andsurrounding sequences. (See, e.g., Triglia, T. et al. (1988) NucleicAcids Res. 16:8186.) A third method, capture PCR, involves PCRamplification of DNA fragments adjacent to known sequences in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to insert anengineered double-stranded sequence into a region of unknown sequencebefore performing PCR. Other methods which may be used to retrieveunknown sequences are known in the art. (See, e.g., Parker, J. D. et al.(1991) Nucleic Acids Res. 19:3055-306). Additionally, one may use PCR,nested primers, and PROMOTERFINDER libraries to walk genomic DNA(Clontech, Palo Alto, Calif.). This procedure avoids the need to screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO 4.06 Primer Analysis software (NationalBiosciences Inc., Plymouth, Minn.) or another appropriate program, to beabout 22 to 30 nucleotides in length, to have a GC content of about 50%or more, and to anneal to the template at temperatures of about 68° C.to 72° C.

[0087] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Inaddition, random-primed libraries, which often include sequencescontaining the 5′ regions of genes, are preferable for situations inwhich an oligo d(T) library does not yield a full-length cDNA. Genomiclibraries may be useful for extension of sequence into 5′non-transcribed regulatory regions.

[0088] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different nucleotide-specific, laser-stimulated fluorescent dyes,and a charge coupled device camera for detection of the emittedwavelengths. Output/light intensity may be converted to electricalsignal using appropriate software (e.g., GENOTYPER and SEQUENCENAVIGATOR, Applied Biosystems), and the entire process from loading ofsamples to computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable forsequencing small DNA fragments which may be present in limited amountsin a particular sample.

[0089] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode hChk1 may be cloned in recombinant DNAmolecules that direct expression of hChk1, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and used to express hChk1.

[0090] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterhChk1-encoding sequences for a variety of purposes including, but notlimited to, modification of the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example,oligonucleotide-mediated site-directed mutagenesis may be used tointroduce mutations that create new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, and so forth.

[0091] In another embodiment, sequences encoding hChk1 may besynthesized, in whole or in part, using chemical methods well known inthe art. (See, e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Symp.Ser. (7)215-223, and Horn, T. et al. (1980) Nucl. Acids. Symp. Ser.(7)225-232.) Alternatively, hChk1 itself or a fragment thereof may besynthesized using chemical methods. For example, peptide synthesis canbe performed using various solid-phase techniques. (See, e.g., Roberge,J. Y. et al. (1995) Science 269:202-204.) Automated synthesis may beachieved using the ABI 431A peptide synthesizer (ABI). Additionally, theamino acid sequence of hChk1, or any part thereof, may be altered duringdirect synthesis and/or combined with sequences from other proteins, orany part thereof, to produce a variant polypeptide.

[0092] The peptide may be substantially purified by preparative highperformance liquid chromatography. (See, e.g, Chiez, R. M. and F. Z.Regnier (1990) Methods Enzymol. 182:392-421.) The composition of thesynthetic peptides may be confirmed by amino acid analysis or bysequencing. (See, e.g., Creighton, T. (1984) Proteins, Structures andMolecular Properties, W H Freeman and Co., New York, N.Y.)

[0093] In order to express a biologically active hChk1, the nucleotidesequences encoding hChk1 or derivatives thereof may be inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for transcriptional and translational control of theinserted coding sequence in a suitable host. These elements includeregulatory sequences, such as enhancers, constitutive and induciblepromoters, and 5′ and 3′ untranslated regions in the vector and inpolynucleotide sequences encoding hChk1. Such elements may vary in theirstrength and specificity. Specific initiation signals may also be usedto achieve more efficient translation of sequences encoding hChk1. Suchsignals include the ATG initiation codon and adjacent sequences, e.g.the Kozak sequence. In cases where sequences encoding hChk1 and itsinitiation codon and upstream regulatory sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including an in-frame ATG initiation codonshould be provided by the vector. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular host cell system used. (See,e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

[0094] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding hChk1and appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning A Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y., ch. 4, 8, and 16-17; and Ausubel, F. M. et al.(1995, and periodic supplements) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y., ch. 9, 13, and 16.)

[0095] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding hChk1. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

[0096] In bacterial systems, a number of cloning and expression vectorsmay be selected depending upon the use intended for polynucleotidesequences encoding hChk1. For example, routine cloning, subcloning, andpropagation of polynucleotide sequences encoding hChk1 can be achievedusing a multifunctional E. coli vector such as Bluescript® (Stratagene)or pSport1™ plasmid (GIBCO BRL). Ligation of sequences encoding hChk1into the vector's multiple cloning site disrupts the lacZ gene, allowinga colorimetric screening procedure for identification of transformedbacteria containing recombinant molecules. In addition, these vectorsmay be useful for in vitro transcription, dideoxy sequencing, singlestrand rescue with helper phage, and creation of nested deletions in thecloned sequence. (See, e.g., Van Heeke, G. and S. M. Schuster (1989) J.Biol. Chem. 264:5503-5509.) When large quantities of hChk1 are needed,e.g. for the production of antibodies, vectors which direct high levelexpression of hChk1 may be used. For example, vectors containing thestrong, inducible T5 or T7 bacteriophage promoter may be used.

[0097] Yeast expression systems may be used for production of hChk1. Anumber of vectors containing constitutive or inducible promoters, suchas alpha factor, alcohol oxidase, and PGH, may be used in the yeastSaccharomyces cerevisiae or Pichia pastoris. In addition, such vectorsdirect either the secretion or intracellular retention of expressedproteins and enable integration of foreign sequences into the hostgenome for stable propagation. (See, e.g., Ausubel, supra; and Grant etal. (1987) Methods Enzymol. 153:516-54; Scorer, C. A. et al. (1994)Bio/Technology 12:181-184.)

[0098] Plant systems may also be used for expression of hChk1.Transcription of sequences encoding hChk1 may be driven viral promoters,e.g., the 35S and 19S promoters of CaMV used alone or in combinationwith the omega leader sequence from TMV. (Takamatsu, N. (1987) EMBO J.6:307-311.) Alternatively, plant promoters such as the small subunit ofRUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. etal. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ.17:85-105.) These constructs can be introduced into plant cells bydirect DNA transformation or pathogen-mediated transfection. (See, e.g.,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.)

[0099] In mammalian cells, a number of viral-based expression systemsmay be utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding hChk1 may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses hChk1 in host cells. (See, e.g., Logan, J. and T. Shenk (1984)Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcriptionenhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used toincrease expression in mammalian host cells. SV40 or EBV-based vectorsmay also be used for high-level protein expression.

[0100] Human artificial chromosomes (HACs) may also be employed todeliver larger fragments of DNA than can be contained in and expressedfrom a plasmid. HACs of about 6 kb to 10 Mb are constructed anddelivered via conventional delivery methods (liposomes, polycationicamino polymers, or vesicles) for therapeutic purposes.

[0101] For long term production of recombinant proteins in mammaliansystems, stable expression of hChk1 in cell lines is preferred. Forexample, sequences encoding hChk1 can be transformed into cell linesusing expression vectors which may contain viral origins of replicationand/or endogenous expression elements and a selectable marker gene onthe same or on a separate vector. Following the introduction of thevector, cells may be allowed to grow for about 1 to 2 days in enrichedmedia before being switched to selective media. The purpose of theselectable marker is to confer resistance to a selective agent, and itspresence allows growth and recovery of cells which successfully expressthe introduced sequences. Resistant clones of stably transformed cellsmay be propagated using tissue culture techniques appropriate to thecell type.

[0102] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase and adeninephosphoribosyltransferase genes, for use in tk⁻ or apr⁻ cells,respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232; andLowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite,antibiotic, or herbicide resistance can be used as the basis forselection. For example, dhfr confers resistance to methotrexate; neoconfers resistance to the aminoglycosides neomycin and G-418; and als orpat confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980)Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin, F. et al (1981) J.Mol. Biol. 150:1-14; and Murry, supra.) Additional selectable genes havebeen described, e.g., trpB and hisD, which alter cellular requirementsfor metabolites. (See, e.g., Hartman, S. C. and R. C. Mulligan (1988)Proc. Natl. Acad. Sci. 85:8047-8051.) Visible markers, e.g.,anthocyanins, green fluorescent proteins (GFP) (Clontech, Palo Alto,Calif.), B glucuronidase and its substrate B-D-glucuronoside, orluciferase and its substrate luciferin may be used. These markers can beused not only to identify transformants, but also to quantify the amountof transient or stable protein expression attributable to a specificvector system. (See, e.g., Rhodes, C. A. et al. (1995) Methods Mol.Biol. 55:121-131.)

[0103] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, the presence and expressionof the gene may need to be confirmed. For example, if the sequenceencoding hChk1 is inserted within a marker gene sequence, transformedcells containing sequences encoding hChk1 can be identified by theabsence of marker gene function. Alternatively, a marker gene can beplaced in tandem with a sequence encoding hChk1 under the control of asingle promoter. Expression of the marker gene in response to inductionor selection usually indicates expression of the tandem gene as well.

[0104] In general, host cells that contain the nucleic acid sequenceencoding hChk1 and that express hChk1 may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCRamplification, and protein bioassay or immunoassay techniques whichinclude membrane, solution, or chip based technologies for the detectionand/or quantification of nucleic acid or protein sequences.

[0105] Immunological methods for detecting and measuring the expressionof hChk1 using either specific polyclonal or monoclonal antibodies areknown in the art. Examples of such techniques include enzyme-linkedimmunosorbent assays (ELISAs), radioimmunoassays (RIAs), andfluorescence activated cell sorting (FACS). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on hChk1 is preferred, but a competitivebinding assay may be employed. These and other assays are well known inthe art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, aLaboratory Manual, APS Press, St Paul, Minn., Section IV; Coligan, J. E.et al. (1997 and periodic supplements) Current Protocols in Immunology,Greene Pub. Associates and Wiley-Interscience, New York, N.Y.; andMaddox, D. E. et al. (1983) J. Exp. Med. 158:1211-1216).

[0106] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding hChk1include oligolabeling, nick translation, end-labeling, or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding hChk1, or any fragments thereof, may be cloned into a vectorfor the production of an mRNA probe. Such vectors are known in the art,are commercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits, such as those provided byPharmacia & Upjohn (Kalamazoo, Mich.), Promega (Madison, Wis.), and U.S.Biochemical Corp. (Cleveland, Ohio). Suitable reporter molecules orlabels which may be used for ease of detection include radionuclides,enzymes, fluorescent, chemiluminescent, or chromogenic agents, as wellas substrates, cofactors, inhibitors, magnetic particles, and the like.

[0107] Host cells transformed with nucleotide sequences encoding hChk1may be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by atransformed cell may be secreted or retained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode hChk1 may be designed to contain signal sequences which directsecretion of hChk1 through a prokaryotic or eukaryotic cell membrane.

[0108] In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to specify protein targeting, folding, and/oractivity. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and W138), are available from the American TypeCulture Collection (ATCC, Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

[0109] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding hChk1 may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric hChk1protein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of hChk1 activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and6-His enable purification of their cognate fusion proteins onimmobilized glutathione, maltose, phenylarsine oxide, calmodulin, andmetal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA)enable immunoaffinity purification of fusion proteins using commerciallyavailable monoclonal and polyclonal antibodies that specificallyrecognize these epitope tags. A fusion protein may also be engineered tocontain a proteolytic cleavage site located between the hChk1 encodingsequence and the heterologous protein sequence, so that hChk1 may becleaved away from the heterologous moiety following purification.Methods for fusion protein expression and purification are discussed inAusubel, F. M. et al. (1995 and periodic supplements) Current Protocolsin Molecular Biology, John Wiley & Sons, New York, N.Y., ch 10. Avariety of commercially available kits may also be used to facilitateexpression and purification of fusion proteins.

[0110] In a further embodiment of the invention, synthesis ofradiolabeled hChk1 may be achieved in vitro using the TNT™ rabbitreticulocyte lysate or wheat germ extract systems (Promega, Madison,Wis.). These systems couple transcription and translation ofprotein-coding sequences operably associated with the T7, T3, or SP6promoters. Translation takes place in the presence of a radiolabeledamino acid precursor, preferably ³⁵S-methionine.

[0111] Fragments of hChk1 may be produced not only by recombinantproduction, but also by direct peptide synthesis using solid-phasetechniques. (See, e.g., Creighton, supra pp. 55-60.) Protein synthesismay be performed by manual techniques or by automation. Automatedsynthesis may be achieved, for example, using the Applied Biosystems431A Peptide Synthesizer (Applied Biosystems). Various fragments ofhChk1 may be synthesized separately and then combined to produce thefull length molecule.

[0112] Therapeutics

[0113] Chemical and structural similarity, e.g., in the context ofsequences and motifs, exists between hChk1 and checkpoint kinase fromyeast (GI 311176). In addition, hChk1 is expressed in cancer andimmortalized cell lines and in tissues associated with the immuneresponse. Therefore, hChk1 appears to play a role in cancer and immunedisorders.

[0114] Therefore, in one embodiment, hChk1 or a fragment or derivativethereof may be administered to a subject to treat or prevent a cancer.Such cancers can include, but are not limited to, adenocarcinoma,leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma,and, in particular, cancers of the adrenal gland, bladder, bone, bonemarrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinaltract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid,penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid,and uterus.

[0115] In another embodiment, a vector capable of expressing hChk1 or afragment or derivative thereof may be administered to a subject to treator prevent a cancer including, but not limited to, those describedabove.

[0116] In a further embodiment, a pharmaceutical composition comprisinga substantially purified hChk1 in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a cancer including, but not limited to, those provided above.

[0117] In still another embodiment, an agonist which modulates theactivity of hChk1 may be administered to a subject to treat or prevent acancer including, but not limited to, those listed above.

[0118] In another embodiment, hChk1 or a fragment or derivative thereofmay be administered to a subject to treat or prevent an immune disorder.Such disorders can include, but are not limited to, acquiredimmunodeficiency syndrome (AIDS), Addison's disease, adult respiratorydistress syndrome, allergies, ankylosing spondylitis, amyloidosis,anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmunethyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn'sdisease, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosisfetalis, erythema nodosum, atrophic gastritis, glomerulonephritis,Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis,hypereosinophilia, irritable bowel syndrome, multiple sclerosis,myasthenia gravis, myocardial or pericardial inflammation,osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis,Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren'ssyndrome, systemic anaphylaxis, systemic lupus erythematosus, systemicsclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Wernersyndrome, complications of cancer, hemodialysis, and extracorporealcirculation, viral, bacterial, fungal, parasitic, protozoal, andhelminthic infections, and trauma.

[0119] In another embodiment, a vector capable of expressing hChk1 or afragment or derivative thereof may be administered to a subject to treator prevent an immune disorder including, but not limited to, thosedescribed above.

[0120] In a further embodiment, a pharmaceutical composition comprisinga substantially purified hChk1 in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent an immune disorder including, but not limited to, those providedabove.

[0121] In still another embodiment, an agonist which modulates theactivity of hChk1 may be administered to a subject to treat or preventan immune disorder including, but not limited to, those listed above.

[0122] In a further embodiment, an antagonist or inhibitor of hChk1 maybe administered to a subject, either alone or in combination withradiation therapy or chemotherapy, to treat a cancer. Such a cancer mayinclude, but is not limited to, those discussed above. In one aspect, anantibody which specifically binds hChk1 may be used directly as anantagonist or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express hChk1.

[0123] In an additional embodiment, a vector expressing a variant ofhChk1 in which the ATP binding residue, K38, or the catalytic siteresidue, D130, are mutated to another amino acid to produce a dominantlyacting, negative regulator of hChk1, may be administered to a subject,either alone or in combination with radiation therapy or chemotherapy,to treat a cancer including, but not limited to, those described above.

[0124] In other embodiments, any of the proteins, antagonists,antibodies, agonists, complementary sequences, or vectors of theinvention may be administered in combination with other appropriatetherapeutic agents. Selection of the appropriate agents for use incombination therapy may be made by one of ordinary skill in the art,according to conventional pharmaceutical principles. The combination oftherapeutic agents may act synergistically to effect the treatment orprevention of the various disorders described above. Using thisapproach, one may be able to achieve therapeutic efficacy with lowerdosages of each agent, thus reducing the potential for adverse sideeffects.

[0125] An antagonist of hChk1 may be produced using methods which aregenerally known in the art. In particular, purified hChk1 may be used toproduce antibodies or to screen libraries of pharmaceutical agents toidentify those which specifically bind hChk1. Antibodies to hChk1 mayalso be generated using methods that are well known in the art. Suchantibodies may include, but are not limited to, polyclonal, monoclonal,chimeric, and single chain antibodies, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies (i.e.,those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0126] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith hChk1 or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

[0127] It is preferred that the oligopeptides, peptides, or fragmentsused to induce antibodies to hChk1 have an amino acid sequenceconsisting of at least about 5 amino acids, and, more preferably, of atleast about 10 amino acids. It is also preferable that theseoligopeptides, peptides, or fragments are identical to a portion of theamino acid sequence of the natural protein and contain the entire aminoacid sequence of a small, naturally occurring molecule. Short stretchesof hChk1 amino acids may be fused with those of another protein, such asKLH, and antibodies to the chimeric molecule may be produced.

[0128] Monoclonal antibodies to hChk1 may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42;Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole,S. P. et al. (1984) Mol. Cell Biol. 62:109-120.)

[0129] In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce hChk1-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci.88:10134-10137.)

[0130] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening immunoglobulin libraries orpanels of highly specific binding reagents as disclosed in theliterature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.86: 3833-3837; and Winter, G. et al. (1991) Nature 349:293-299.)

[0131] Antibody fragments which contain specific binding sites for hChk1may also be generated. For example, such fragments include, but are notlimited to, F(ab′)2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huse,W. D. et al. (1989) Science 246:1275-1281.)

[0132] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between hChk1 and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering hChk1 epitopes is preferred, but a competitivebinding assay may also be employed. (Maddox, supra.)

[0133] In another embodiment of the invention, the polynucleotidesencoding hChk1, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding hChk1 may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding hChk1. Thus, complementary molecules orfragments may be used to modulate hChk1 activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligonucleotides or larger fragments can bedesigned from various locations along the coding or control regions ofsequences encoding hChk1.

[0134] Expression vectors derived from retroviruses, adenoviruses, orherpes or vaccinia viruses, or from various bacterial plasmids, may beused for delivery of nucleotide sequences to the targeted organ, tissue,or cell population. Methods which are well known to those skilled in theart can be used to construct vectors to express nucleic acid sequencescomplementary to the polynucleotides encoding hChk1. (See, e.g.,Sambrook, supra; and Ausubel, supra.)

[0135] Genes encoding hChk1 can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide, or fragment thereof, encoding hChk1. Such constructs maybe used to introduce untranslatable sense or antisense sequences into acell. Even in the absence of integration into the DNA, such vectors maycontinue to transcribe RNA molecules until they are disabled byendogenous nucleases. Transient expression may last for a month or morewith a non-replicating vector, and may last even longer if appropriatereplication elements are part of the vector system.

[0136] As mentioned above, modifications of gene expression can beobtained by designing complementary sequences or antisense molecules(DNA, RNA, or PNA) to the control, 5′, or regulatory regions of the geneencoding hChk1. Oligonucleotides derived from the transcriptioninitiation site, e.g., between about positions −10 and +10 from thestart site, are preferred. Similarly, inhibition can be achieved usingtriple helix base-pairing methodology. Triple helix pairing is usefulbecause it causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (See, e.g., Gee, J. E. et al. (1994)in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches,Futura Publishing Co., Mt. Kisco, N.Y., pp. 163-177.) A complementarysequence or antisense molecule may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

[0137] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodinghChk1.

[0138] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0139] Complementary ribonucleic acid molecules and ribozymes of theinvention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. These include techniques forchemically synthesizing oligonucleotides such as solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding hChk1. Such DNA sequences may be incorporated into a widevariety of vectors with suitable RNA polymerase promoters such as T7 orSP6. Alternatively, these cDNA constructs that synthesize complementaryRNA, constitutively or inducibly, can be introduced into cell lines,cells, or tissues.

[0140] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule, or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0141] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection, by liposomeinjections, or by polycationic amino polymers may be achieved usingmethods which are well known in the art. (See, e.g., Goldman, C. K. etal. (1997) Nature Biotechnology 15:462-466.)

[0142] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0143] An additional embodiment of the invention relates to theadministration of a pharmaceutical or sterile composition, inconjunction with a pharmaceutically acceptable carrier, for any of thetherapeutic effects discussed above. Such pharmaceutical compositionsmay consist of hChk1, antibodies to hChk1, and mimetics, agonists,antagonists, or inhibitors of hChk1. The compositions may beadministered alone or in combination with at least one other agent, suchas a stabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier including, but not limited to,saline, buffered saline, dextrose, and water. The compositions may beadministered to a patient alone, or in combination with other agents,drugs, or hormones.

[0144] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0145] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0146] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0147] Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

[0148] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0149] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with fillers or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0150] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils, such as sesame oil, orsynthetic fatty acid esters, such as ethyl oleate, triglycerides, orliposomes. Non-lipid polycationic amino polymers may also be used fordelivery. Optionally, the suspension may also contain suitablestabilizers or agents to increase the solubility of the compounds andallow for the preparation of highly concentrated solutions.

[0151] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0152] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0153] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tendto be more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

[0154] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of hChk1, such labeling wouldinclude amount, frequency, and method of administration.

[0155] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0156] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells or in animal models such as mice, rats, rabbits, dogs, or pigs. Ananimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0157] A therapeutically effective dose refers to that amount of activeingredient, for example hChk1 or fragments thereof, antibodies of hChk1,and agonists, antagonists or inhibitors of hChk1, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, and it can be expressed asthe LD₅₀/ED₅₀ ratio. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies are used to formulate a range of dosage forhuman use. The dosage contained in such compositions is preferablywithin a range of circulating concentrations that includes the ED₅₀ withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, the sensitivity of the patient, and theroute of administration.

[0158] The exact dosage will be determined by the practitioner, in lightof factors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, orbiweekly depending on the half-life and clearance rate of the particularformulation.

[0159] Normal dosage amounts may vary from about 0.1 μg to 100,000 μg,up to a total dose of about 1 gram, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0160] Diagnostics

[0161] In another embodiment, antibodies which specifically bind hChk1may be used for the diagnosis of disorders characterized by expressionof hChk1, or in assays to monitor patients being treated with hChk1 oragonists, antagonists, or inhibitors of hChk1. Antibodies useful fordiagnostic purposes may be prepared in the same manner as describedabove for therapeutics. Diagnostic assays for hChk1 include methodswhich utilize the antibody and a label to detect hChk1 in human bodyfluids or in extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by covalent ornon-covalent attachment of a reporter molecule. A wide variety ofreporter molecules, several of which are described above, are known inthe art and may be used.

[0162] A variety of protocols for measuring hChk1, including ELISAs,RIAs, and FACS, are known in the art and provide a basis for diagnosingaltered or abnormal levels of hChk1 expression. Normal or standardvalues for hChk1 expression are established by combining body fluids orcell extracts taken from normal mammalian subjects, preferably human,with antibody to hChk1 under conditions suitable for complex formation.The amount of standard complex formation may be quantitated by variousmethods, preferably by photometric means. Quantities of hChk1 expressedin subject samples, control and disease, from biopsied tissues arecompared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0163] In another embodiment of the invention, the polynucleotidesencoding hChk1 may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, complementary RNAand DNA molecules, and PNAs. The polynucleotides may be used to detectand quantitate gene expression in biopsied tissues in which expressionof hChk1 may be correlated with disease. The diagnostic assay may beused to determine absence, presence, and excess expression of hChk1, andto monitor regulation of hChk1 levels during therapeutic intervention.

[0164] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding hChk1 or closely related molecules may be used to identifynucleic acid sequences which encode hChk1. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5′regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding hChk1, allelicvariants, or related sequences.

[0165] Probes may also be used for the detection of related sequences,and should preferably have at least 50% sequence identity to any of thehChk1 encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and may be derived from the sequence of SEQID NO:2 or from genomic sequences including promoters, enhancers, andintrons of the hChk1 gene.

[0166] Means for producing specific hybridization probes for DNAsencoding hChk1 include the cloning of polynucleotide sequences encodinghChk1 or hChk1 derivatives into vectors for the production of mRNAprobes. Such vectors are known in the art, are commercially available,and may be used to synthesize RNA probes in vitro by means of theaddition of the appropriate RNA polymerases and the appropriate labelednucleotides. Hybridization probes may be labeled by a variety ofreporter groups, for example, by radionuclides such as ³²P or 35S, or byenzymatic labels, such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems, and the like.

[0167] Polynucleotide sequences encoding hChk1 may be used for thediagnosis of a disorder associated with expression of hChk1. Examples ofsuch a disorder include, but are not limited to, cancers, such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, andteratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus, and immune disorders suchas acquired immunodeficiency syndrome (AIDS), Addison's disease, adultrespiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, bronchitis, cholecystitis, contactdermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, episodic lymphopenia withlymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowelsyndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, Sjögren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerativecolitis, uveitis, Werner syndrome, complications of cancer,hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,parasitic, protozoal, and helminthic infections, and trauma. Thepolynucleotide sequences encoding hChk1 may be used in Southern ornorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; in dipstick, pin, and ELISA assays; and in microarraysutilizing fluids or tissues from patients to detect altered hChk1expression. Such qualitative or quantitative methods are well known inthe art.

[0168] In a particular aspect, the nucleotide sequences encoding hChk1may be useful in assays that detect the presence of associateddisorders, particularly those mentioned above. The nucleotide sequencesencoding hChk1 may be labeled by standard methods and added to a fluidor tissue sample from a patient under conditions suitable for theformation of hybridization complexes. After a suitable incubationperiod, the sample is washed and the signal is quantitated and comparedwith a standard value. If the amount of signal in the patient sample issignificantly altered in comparison to a control sample then thepresence of altered levels of nucleotide sequences encoding hChk1 in thesample indicates the presence of the associated disorder. Such assaysmay also be used to evaluate the efficacy of a particular therapeutictreatment regimen in animal studies, in clinical trials, or to monitorthe treatment of an individual patient.

[0169] In order to provide a basis for the diagnosis of a disorderassociated with expression of hChk1, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, encoding hChk1, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with values from an experiment in which a known amountof a substantially purified polynucleotide is used. Standard valuesobtained in this manner may be compared with values obtained fromsamples from patients who are symptomatic for a disorder. Deviation fromstandard values is used to establish the presence of a disorder.

[0170] Once the presence of a disorder is established and a treatmentprotocol is initiated, hybridization assays may be repeated on a regularbasis to determine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0171] With respect to cancer, the presence of a relatively high amountof transcript, or an abnormally low amount, in biopsied tissue from anindividual may indicate a predisposition for the development of thedisease, or may provide a means for detecting the disease prior to theappearance of actual clinical symptoms. A more definitive diagnosis ofthis type may allow health professionals to employ preventative measuresor aggressive treatment earlier thereby preventing the development orfurther progression of the cancer.

[0172] Additional diagnostic uses for oligonucleotides designed from thesequences encoding hChk1 may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding hChk1, or a fragment of a polynucleotide complementary to thepolynucleotide encoding hChk1, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

[0173] Methods which may also be used to quantitate the expression ofhChk1 include radiolabeling or biotinylating nucleotides,coamplification of a control nucleic acid, and interpolating resultsfrom standard curves. (See, e.g., Melby, P. C. et al. (1993) J. Immunol.Methods 159:235-244; and Duplaa, C. et al. (1993) Anal. Biochem.212:229-236.) The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor calorimetric response gives rapid quantitation.

[0174] In further embodiments, oligonucleotides or longer fragmentsderived from any of the polynucleotide sequences described herein may beused as targets in a microarray. The microarray can be used to monitorthe expression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents.

[0175] Microarrays may be prepared, used, and analyzed using methodsknown in the art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci.93:10614-10619; Baldeschweiler et al. (1995) PCT applicationWO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; andHeller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)

[0176] In another embodiment of the invention, nucleic acid sequencesencoding hChk1 may be used to generate hybridization probes useful inmapping the naturally occurring genomic sequence. The sequences may bemapped to a particular chromosome, to a specific region of a chromosome,or to artificial chromosome constructions, e.g., human artificialchromosomes (HACs), yeast artificial chromosomes (YACs), bacterialartificial chromosomes (BACs), bacterial PI constructions, or singlechromosome cDNA libraries. (See, e.g., Price, C. M. (1993) Blood Rev.7:127-134; and Trask, B. J. (1991) Trends Genet. 7:149-154.)

[0177] Fluorescent in situ hybridization (FISH) may be correlated withother physical chromosome mapping techniques and genetic map data. (See,e.g., Heinz-Ulrich, et al. (1995) in Meyers, R. A. (ed.) MolecularBiology and Biotechnology, VCH Publishers New York, N.Y., pp. 965-968.)Examples of genetic map data can be found in various scientific journalsor at the Online Mendelian Inheritance in Man (OMIM) site. Correlationbetween the location of the gene encoding hChk1 on a physicalchromosomal map and a specific disorder, or a predisposition to aspecific disorder, may help define the region of DNA associated withthat disorder. The nucleotide sequences of the invention may be used todetect differences in gene sequences among normal, carrier, and affectedindividuals.

[0178] In situ hybridization of chromosomal preparations and physicalmapping techniques, such as linkage analysis using establishedchromosomal markers, may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms by physical mapping. This provides valuableinformation to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, e.g., ataxia-telangiectasia to 11q22-23, anysequences mapping to that area may represent associated or regulatorygenes for further investigation. (See, e.g., Gatti, R. A. et al. (1988)Nature 336:577-580.) The nucleotide sequence of the subject inventionmay also be used to detect differences in the chromosomal location dueto translocation, inversion, etc., among normal, carrier, or affectedindividuals.

[0179] In another embodiment of the invention, hChk1, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes betweenhChk1 and the agent being tested may be measured.

[0180] Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationWO84/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The test compounds are reacted with hChk1, orfragments thereof, and washed. Bound hChk1 is then detected by methodswell known in the art. Purified hChk1 can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

[0181] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding hChk1specifically compete with a test compound for binding hChk1. In thismanner, antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with hChk1.

[0182] In additional embodiments, the nucleotide sequences which encodehChk1 may be used in any molecular biology techniques that have yet tobe developed, provided the new techniques rely on properties ofnucleotide sequences that are currently known, including, but notlimited to, such properties as the triplet genetic code and specificbase pair interactions.

[0183] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0184] I. MMLR1DT01 cDNA Library Construction

[0185] The normal peripheral blood macrophages used for this librarywere obtained from two 24 year old, Caucasian males. The MMLR1DT01library represents a mixture of allogeneically stimulated humanmacrophage populations obtained from Ficoll/Hypaque purified buffycoats. The cells from the two different donors (not typed for HLAalleles) were incubated at a density of 1×10⁶/ml for 48 hours in DMEcontaining 10% human serum.

[0186] After incubation, macrophages mostly adhered to the plasticsurface of the petri dish, and most other cell types, B and Tlymphocytes, remained in solution. The DME was decanted from the wells,and the wells were washed with phosphate buffered saline (PBS).Macrophages were released from the plastic surface by gently scrapingthe petri dishes in PBS/1 mM EDTA. Macrophages were lysed immediately inbuffer containing guanidinium isothiocyanate.

[0187] The lysate was extracted twice with a mixture of acid phenol pH4.0 and centrifuged over a CsCl cushion using a Beckman SW28 rotor in aL8-70M Ultracentrifuge (Beckman Instruments, Fullerton Calif.). The RNAwas precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,resuspended in water and treated with DNase for 15 min at 37° C. It mustbe noted that some contaminating T and B lymphocytes may have beenpresent.

[0188] The RNA was used in the SUPERSCRIPT Plasmid System for cDNAsynthesis and plasmid cloning (Life Technologies, Gaithersburg Md.) withthe recommended protocol. cDNAs were fractionated on a Sepharose CL4Bcolumn (catalog #275105, Pharmacia Biotech, Pistcataway, N.J.) and thosecDNAs exceeding 400 bp were ligated into PSPORT 1. The plasmid wastransformed into chemically competent DH5α host cells (LifeTechnologies).

[0189] II. Isolation and Sequencing of cDNA Clones

[0190] Plasmid DNA was released from the cells and purified using theMINIPREP Kit (Edge Biosystems, Gaithersburg Md.). The recommendedprotocol was employed except for the following changes: 1) the 96 wellswere each filled with only 1 ml of sterile TERRIFIC BROTH (LifeTechnologies) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) thebacteria were cultured for 24 hours after the wells were inoculated andthen lysed with 60 μl of lysis buffer; 3) a centrifugation stepemploying the Beckman GS-6R @2900 rpm for 5 min was performed before thecontents of the block were added to the primary filter plate; and 4) theoptional step of adding isopropanol to TRIS buffer was not routinelyperformed. After the last step in the protocol, samples were transferredto a Beckman 96-well block for storage.

[0191] The cDNAs were prepared using a Hamilton Micro Lab 2200(Hamilton, Reno Nev.) in combination with four Peltier Thermal Cyclers(PTC200 from MJ Research, Watertown Mass.). The cDNAs were sequenced bythe method of Sanger F and AR Coulson (1975; J Mol Biol 94:441f) usingABI 377 or 373 DNA Sequencing Systems (Applied Biosystems), and readingframe was determined.

[0192] III. Analysis of cDNA Clones and Their Deduced Proteins

[0193] The cDNA sequences and the full length nucleotide and amino acidsequences disclosed in the Sequence Listing were queried againstdatabases such as GenBank primate (pri), rodent (rod), mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp) databases, SwissProt, BLOCKS,and other databases which contain previously identified and annotatedmotifs and sequences. Algorithms such as Smith Waterman which deal withprimary sequence patterns and secondary structure gap penalties (Smith,T. et al. (1992) Protein Engineering 5:35-51) and programs andalgorithms such as BLAST (Basic Local Alignment Search Tool; Altschul,S.F. (1993) J. Mol. Evol 36:290-300; and Altschul et al. (1990) J. Mol.Biol. 215:403-410), and HMM (Hidden Markov Models; Eddy, S.R. (1996)Cur. Opin. Str. Biol. 6:361-365 and Sonnhammer, E. L. L. et al. (1997)Proteins 28:405-420) were used to assemble and analyze nucleotide andamino acid sequences. The databases, programs, algorithms, methods andtools are available, well known in the art, and described in Ausubel(supra, unit 7.7), in Meyers, R. A. (1995; Molecular Biology andBiotechnology, Wiley VCH, Inc, New York N.Y., p 856-853), indocumentation provided with software (Genetics Computer Group (GCG),Madison Wis.), and on the world wide web (www). Two comprehensivewebsites which list, describe, and/or link many of the databases andtools are: 1) the www resource in practical sequence analysis(http://genome.wustl.edu/eddy/bio5495/online_resources.html), and 2) thebibliography of computational gene recognition(http://linkage.rockefeller.edu/wli/gene/programs.html). For example,the first website links PFAM as a database(http://genome.wustl.edu/Pfam/) and as an HMM search tool(http://genome.wustl.edu/eddy/cgi-bin/hmm_page.cgi).

[0194] Table 1 summarizes the databases and tools used herein. The firstcolumn of Table 1 shows the tool, program, or algorithm; the secondcolumn, the database; the third column, a brief description; and thefourth column (where applicable), scores for determining the strength ofa match between two sequences (the higher the value, the morehomologous).

[0195] IV. Northern Analysis

[0196] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound. (See, e.g., Sambrook,supra, ch. 7; and Ausubel, supra, ch. 4 and 16.)

[0197] Analogous computer techniques applying BLAST were used to searchfor identical or related molecules in databases such as GenBank orLIFESEQ database (Incyte Genomics). This analysis is much faster thanmultiple membrane-based hybridizations. In addition, the sensitivity ofthe computer search can be modified to determine whether any particularmatch is categorized as exact or similar.

[0198] The basis of the search is the product score, which is definedas:$\frac{\% \quad {sequence}\quad {identity} \times \% \quad {maximum}\quad {BLAST}\quad {score}}{100}$

[0199] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1% to 2% error, and, with a product score of 70, the match will beexact. Similar molecules are usually identified by selecting those whichshow product scores between 15 and 40, although lower scores mayidentify related molecules.

[0200] The results of Northern analysis are shown in FIG. 4, giving thelist of libraries in which the transcript encoding hChk1 was found.Abundance and percent abundance are also reported. Abundance directlyreflects the number of times a particular transcript is represented in acDNA library, and percent abundance is abundance divided by the totalnumber of sequences examined in the cDNA library.

[0201] V. Extension of hChk1 Encoding Polynucleotides

[0202] Oligonucleotide primers were designed from clones 516219 and2044650 for PCR to enable full length construction of hChk1.Oligonucleotide 1 (5′ GTGAATTCACCACCATGGCAGTGCCCTTTGTGGAAGACTGGGACTTGGTGCAAACCCTGGGAGAAGGTGCCTATGGAGAAGTTCAA C 3′; SEQID NO:13) was extended by PCR to complete the 5′ end of cDNA clone2044650 inclusive of the ATG start codon (in the context of a kozakconsensus sequence) and an EcoR1 restriction site. Oligonucleotide 2 (5′CACTCGAGGATTCCCCAGAGCCGATGGTC 3′; SEQ ID NO:14) was extended by PCR tocomplete the 3′ end of cDNA clone 516219 inclusive of the TGA stop codonand an Xho1 restriction site. Oligonucleotide 3 (5′GGAATAACTCACAGGGATATTAAACCAGAAAATCT TCTGTTGGATGAAAGGGATAAC 3′; SEQ IDNO:15), derived from the consensus sequence, was used to bridge the gapbetween clones 516219 and 2044650.

[0203] VI. Labeling and Use of Individual Hybridization Probes

[0204] Hybridization probes derived from SEQ ID NO:2 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [γ-³²P] adenosinetriphosphate (APB), and T4 polynucleotide kinase (DuPont NEN, Boston,Mass.). The labeled oligonucleotides are substantially purified using aSEPHADEX G-25 superfine size exclusion dextran bead column (APB). Analiquot containing 10⁷ counts per minute of the labeled probe is used ina typical membrane-based hybridization analysis of human genomic DNAdigested with one of the following endonucleases: Ase I, Bgl II, Eco RI,Pst I, Xbal, or Pvu II (DuPont NEN,).

[0205] The DNA from each digest is fractionated on a 0.7% agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Eastman Kodak, Rochester, N.Y.) is exposed to the blots to film forseveral hours, hybridization patterns are compared visually.

[0206] VII. Microarrays

[0207] A chemical coupling procedure and an ink jet device can be usedto synthesize array elements on the surface of a substrate. (See, e.g.,Baldeschweiler, supra.) An array analogous to a dot or slot blot mayalso be used to arrange and link elements to the surface of a substrateusing thermal, UV, chemical, or mechanical bonding procedures. A typicalarray may be produced by hand or using available methods and machinesand contain any appropriate number of elements. After hybridization,nonhybridized probes are removed and a scanner used to determine thelevels and patterns of fluorescence. The degree of complementarity andthe relative abundance of each probe which hybridizes to an element onthe microarray may be assessed through analysis of the scanned images.

[0208] Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragmentsthereof may comprise the elements of the microarray. Fragments suitablefor hybridization can be selected using software well known in the artsuch as LASERGENE. Full-length cDNAs, ESTs, or fragments thereofcorresponding to one of the nucleotide sequences of the presentinvention, or selected at random from a cDNA library relevant to thepresent invention, are arranged on an appropriate substrate, e.g., aglass slide. The cDNA is fixed to the slide using, e.g., UVcross-linking followed by thermal and chemical treatments and subsequentdrying. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; andShalon, D. et al. (1996) Genome Res. 6:639-645.) Fluorescent probes areprepared and used for hybridization to the elements on the substrate.The substrate is analyzed by procedures described above.

[0209] VIII. Complementary Polynucleotides

[0210] Sequences complementary to the hChk1-encoding sequences, or anyparts thereof, are used to detect, decrease, or inhibit expression ofnaturally occurring hChk1. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO™ 4.06 software andthe coding sequence of hChk1. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique 5′ sequence and used toprevent promoter binding to the coding sequence. To inhibit translation,a complementary oligonucleotide is designed to prevent ribosomal bindingto the hChk1-encoding transcript.

[0211] IX. Expression of hChk1

[0212] Expression and purification of hChk1 was achieved using variousbacterial, mammalian, or virus-based expression systems. Vectors wereconstructed using both active hChk1 kinase (SEQ ID NO:2) and an inactivekinase (K38A; SEQ ID NO:16) achieved by changing amino acid K38 in SEQID NO:1 to alanine (A38) by nucleotide base changes in SEQ ID NO:2 fromA128 AG to G128CG. Table 2 summarizes the various expression vectors,host cells, and their sources used in the expression of these sequences.TABLE 2 Vector Host Cell Source pGex4T3 E. coli APB pFastBac Insect LifeTechnologies pFastBac HTC Insect Life Technologies pIND MammalianInvitrogen, Carlsbad, CA pcDNA3.1 Mammalian Invitrogen

[0213] hChk1 obtained by these methods were used directly in thefollowing activity assay.

[0214] X. Demonstration of hChk1 Activity

[0215] Protein kinase activity of hChk1 was demonstrated in vitro in anassay containing hChk1, 50 μl of kinase buffer, 1 μg substrate, such asmylein basic protein (MBP) or synthetic peptide substrates, 1 mM DTT, 10μg ATP, and 0.5 μCi [γ-³³P]ATP. The reaction was incubated at 30° C. for30 minutes and stopped by pipetting onto P81 paper. The incorporatedradioactivity was measured using a radioactivity scintillation counter.Alternatively, the reaction was stopped by heating to 100° C. in thepresence of SDS loading buffer and visualized on a 12% SDSpolyacrylamide gel by autoradiography. Incorporated radioactivity wascorrected for reactions carried out in the absence of hChk1 or in thepresence of the inactive kinase, K38A (SEQ ID NO:16).

[0216] In the alternative, other assays for hChk1 might includescintillation proximity assays (SPA), scintillation plate technology andfilter binding assays. Useful substrates include recombinant proteinstagged with glutathione transferase, or synthetic peptide substratestagged with biotin. Inhibitors of hChk1 activity, such as small organicmolecules, proteins or peptides, may be identified by such assays.

[0217] XI. Functional Assays

[0218] hChk1 function is assessed by expressing the sequences encodinghChk1 at physiologically elevated levels in mammalian cell culturesystems. cDNA is subcloned into a mammalian expression vector containinga strong promoter that drives high levels of cDNA expression. Vectors ofchoice include pCMV SPORT (Life Technologies) and pCR 3.1 (Invitrogen),both of which contain the cytomegalovirus promoter. 5-10 μg ofrecombinant vector are transiently transfected into a human cell line,preferably of endothelial or hematopoietic origin, using either liposomeformulations or electroporation. 1-2 μg of an additional plasmidcontaining sequences encoding a marker protein are co-transfected.Expression of a marker protein provides a means to distinguishtransfected cells from nontransfected cells and is a reliable predictorof cDNA expression from the recombinant vector. Marker proteins ofchoice include, e.g., Green Fluorescent Protein (GFP) (Clontech), CD64,or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laseroptics-based technique, is used to identify transfected cells expressingGFP or CD64-GFP, and to evaluate properties, for example, theirapoptotic state. FCM detects and quantifies the uptake of fluorescentmolecules that diagnose events preceding or coincident with cell death.These events include changes in nuclear DNA content as measured bystaining of DNA with propidium iodide; changes in cell size andgranularity as measured by forward light scatter and 90 degree sidelight scatter; down-regulation of DNA synthesis as measured by decreasein bromodeoxyuridine uptake; alterations in expression of cell surfaceand intracellular proteins as measured by reactivity with specificantibodies; and alterations in plasma membrane composition as measuredby the binding of fluorescein-conjugated Annexin V protein to the cellsurface. Methods in flow cytometry are discussed in Ormerod, M. G.(1994) Flow Cytometry, Oxford, New York, N.Y.

[0219] The influence of hChk1 on gene expression can be assessed usinghighly purified populations of cells transfected with sequences encodinghChk1 and either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed onthe surface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Lake Success, N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art. Expression of mRNA encoding hChk1 and other genes of interestcan be analyzed by Northern analysis or microarray techniques.

[0220] XII. Production of hChk1 Specific Antibodies

[0221] hChk1 substantially purified using polyacrylamide gelelectrophoresis (PAGE)(see, e.g., Harrington, M. G. (1990) MethodsEnzymol. 182:488-495), or other purification techniques, is used toimmunize rabbits and to produce antibodies using standard protocols.

[0222] Alternatively, the hChk1 amino acid sequence is analyzed usingLASERGENE software (DNASTAR Inc.) to determine regions of highimmunogenicity, and a corresponding oligopeptide is synthesized and usedto raise antibodies by means known to those of skill in the art. Methodsfor selection of appropriate epitopes, such as those near the C-terminusor in hydrophilic regions are well described in the art. (See, e.g.,Ausubel supra, ch. 11.)

[0223] Typically, oligopeptides 15 residues in length are synthesizedusing an Applied Biosystems peptide synthesizer Model 431A usingfmoc-chemistry and coupled to KLH (Sigma, St. Louis, Mo.) by reactionwith N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity. (See, e.g., Ausubel supra.) Rabbits are immunized withthe oligopeptide-KLH complex in complete Freund's adjuvant. Resultingantisera are tested for antipeptide activity by, for example, bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radio-iodinated goat anti-rabbitIgG.

[0224] XIII. Purification of Naturally Occurring hChk1 Using SpecificAntibodies

[0225] Naturally occurring or recombinant hChk1 is substantiallypurified by immunoaffinity chromatography using antibodies specific forhChk1. An immunoaffinity column is constructed by covalently couplinganti-hChk1 antibody to an activated chromatographic resin, such asCNBr-activated SEPHAROSE (APB). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

[0226] Media containing hChk1 are passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of hChk1 (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/hChk1 binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), andhChk1 is collected.

[0227] XIV. Identification of Molecules Which Interact with hChk1

[0228] hChk1, or biologically active fragments thereof, are labeled with¹²⁵I Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled hChk1, washed, and anywells with labeled hChk1 complex are assayed. Data obtained usingdifferent concentrations of hChk1 are used to calculate values for thenumber, affinity, and association of hChk1 with the candidate molecules.

[0229] Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 16 <210> SEQ ID NO 1<211> LENGTH: 476 <212> TYPE: PRT <213> ORGANISM: HOMO SAPIENS <220>FEATURE: <223> OTHER INFORMATION: 516219, MMLR1DT01 <400> SEQUENCE: 1Met Ala Val Pro Phe Val Glu Asp Trp Asp Leu Val Gln Thr Leu Gly 1 5 1015 Glu Gly Ala Tyr Gly Glu Val Gln Leu Ala Val Asn Arg Val Thr Glu 20 2530 Glu Ala Val Ala Val Lys Ile Val Asp Met Lys Arg Ala Val Asp Cys 35 4045 Pro Glu Asn Ile Lys Lys Glu Ile Cys Ile Asn Lys Met Leu Asn His 50 5560 Glu Asn Val Val Lys Phe Tyr Gly His Arg Arg Glu Gly Asn Ile Gln 65 7075 80 Tyr Leu Phe Leu Glu Tyr Cys Ser Gly Gly Glu Leu Phe Asp Arg Ile 8590 95 Glu Pro Asp Ile Gly Met Pro Glu Pro Asp Ala Gln Arg Phe Phe His100 105 110 Gln Leu Met Ala Gly Val Val Tyr Leu His Gly Ile Gly Ile ThrHis 115 120 125 Arg Asp Ile Lys Pro Glu Asn Leu Leu Leu Asp Glu Arg AspAsn Leu 130 135 140 Lys Ile Ser Asp Phe Gly Leu Ala Thr Val Phe Arg TyrAsn Asn Arg 145 150 155 160 Glu Arg Leu Leu Asn Lys Met Cys Gly Thr LeuPro Tyr Val Ala Pro 165 170 175 Glu Leu Leu Lys Arg Arg Glu Phe His AlaGlu Pro Val Asp Val Trp 180 185 190 Ser Cys Gly Ile Val Leu Thr Ala MetLeu Ala Gly Glu Leu Pro Trp 195 200 205 Asp Gln Pro Ser Asp Ser Cys GlnGlu Tyr Ser Asp Trp Lys Glu Lys 210 215 220 Lys Thr Tyr Leu Asn Pro TrpLys Lys Ile Asp Ser Ala Pro Leu Ala 225 230 235 240 Leu Leu His Lys IleLeu Val Glu Asn Pro Ser Ala Arg Ile Thr Ile 245 250 255 Pro Asp Ile LysLys Asp Arg Trp Tyr Asn Lys Pro Leu Lys Lys Gly 260 265 270 Ala Lys ArgPro Arg Val Thr Ser Gly Gly Val Ser Glu Ser Pro Ser 275 280 285 Gly PheSer Lys His Ile Gln Ser Asn Leu Asp Phe Ser Pro Val Asn 290 295 300 SerAla Ser Ser Glu Glu Asn Val Lys Tyr Ser Ser Ser Gln Pro Glu 305 310 315320 Pro Arg Thr Gly Leu Ser Leu Trp Asp Thr Ser Pro Ser Tyr Ile Asp 325330 335 Lys Leu Val Gln Gly Ile Ser Phe Ser Gln Pro Thr Cys Pro Asp His340 345 350 Met Leu Leu Asn Ser Gln Leu Leu Gly Thr Pro Gly Ser Ser GlnAsn 355 360 365 Pro Trp Gln Arg Leu Val Lys Arg Met Thr Arg Phe Phe ThrLys Leu 370 375 380 Asp Ala Asp Lys Ser Tyr Gln Cys Leu Lys Glu Thr CysGlu Lys Leu 385 390 395 400 Gly Tyr Gln Trp Lys Lys Ser Cys Met Asn GlnVal Thr Ile Ser Thr 405 410 415 Thr Asp Arg Arg Asn Asn Lys Leu Ile PheLys Val Asn Leu Leu Glu 420 425 430 Met Asp Asp Lys Ile Leu Val Asp PheArg Leu Ser Lys Gly Asp Gly 435 440 445 Leu Glu Phe Lys Arg His Phe LeuLys Ile Lys Gly Lys Leu Ile Asp 450 455 460 Ile Val Ser Ser Gln Lys ValTrp Leu Pro Ala Thr 465 470 475 <210> SEQ ID NO 2 <211> LENGTH: 1479<212> TYPE: DNA <213> ORGANISM: HOMO SAPIENS <220> FEATURE: <223> OTHERINFORMATION: 516219, MMLR1DT01 <400> SEQUENCE: 2 gaattcggct tccaccatggcagtgccctt tgtggaagac tgggacttgg tgcaaaccct 60 gggagaaggt gcctatggagaagttcaact tgctgtgaat agagtaactg aagaagcagt 120 cgcagtgaag attgtagatatgaagcgtgc cgtagactgt ccagaaaata ttaagaaaga 180 gatctgtatc aataaaatgctaaatcatga aaatgtagta aaattctatg gtcacaggag 240 agaaggcaat atccaatatttatttctgga gtactgtagt ggaggagagc tttttgacag 300 aatagagcca gacataggcatgcctgaacc agatgctcag agattcttcc atcaactcat 360 ggcaggggtg gtttatctgcatggtattgg aataactcac agggatatta aaccagaaaa 420 tcttctgttg gatgaaagggataacctcaa aatctcagac tttggcttgg caacagtatt 480 tcggtataat aatcgtgagcgtttgttgaa caagatgtgt ggtactttac catatgttgc 540 tccagaactt ctgaagagaagagaatttca tgcagaacca gttgatgttt ggtcctgtgg 600 aatagtactt actgcaatgctcgctggaga attgccatgg gaccaaccca gtgacagctg 660 tcaggagtat tctgactggaaagaaaaaaa aacatacctc aacccttgga aaaaaatcga 720 ttctgctcct ctagctctgctgcataaaat cttagttgag aatccatcag caagaattac 780 cattccagac atcaaaaaagatagatggta caacaaaccc ctcaagaaag gggcaaaaag 840 gccccgagtc acttcaggtggtgtgtcaga gtctcccagt ggattttcta agcacattca 900 atccaatttg gacttctctccagtaaacag tgcttctagt gaagaaaatg tgaagtactc 960 cagttctcag ccagaaccccgcacaggtct ttccttatgg gataccagcc cctcatacat 1020 tgataaattg gtacaagggatcagcttttc ccagcccaca tgtcctgatc atatgctttt 1080 gaatagtcag ttacttggcaccccaggatc ctcacagaac ccctggcagc ggttggtcaa 1140 aagaatgaca cgattctttaccaaattgga tgcagacaaa tcttatcaat gcctgaaaga 1200 gacttgtgag aagttgggctatcaatggaa gaaaagttgt atgaatcagg ttactatatc 1260 aacaactgat aggagaaacaataaactcat tttcaaagtg aatttgttag aaatggatga 1320 taaaatattg gttgacttccggctttctaa gggtgatgga ttggagttca agagacactt 1380 cctgaagatt aaagggaagctgattgatat tgtgagcagc cagaaggttt ggcttcctgc 1440 cacatgatcg gaccatcggctctggggaat cctcgagtg 1479 <210> SEQ ID NO 3 <211> LENGTH: 496 <212>TYPE: PRT <213> ORGANISM: HOMO SAPIENS <220> FEATURE: <223> OTHERINFORMATION: 311176, GenBank <400> SEQUENCE: 3 Met Ala Gln Lys Leu AspAsn Phe Pro Tyr His Ile Gly Arg Glu Ile 1 5 10 15 Gly Thr Gly Ala PheAla Ser Val Arg Leu Cys Tyr Asp Asp Asn Ala 20 25 30 Lys Ile Tyr Ala ValLys Phe Val Asn Lys Lys His Ala Thr Ser Cys 35 40 45 Met Asn Ala Gly ValTrp Ala Arg Arg Met Ala Ser Glu Ile Gln Leu 50 55 60 His Lys Leu Cys AsnGly His Lys Asn Ile Ile His Phe Tyr Asn Thr 65 70 75 80 Ala Glu Asn ProGln Trp Arg Trp Val Val Leu Glu Phe Ala Gln Gly 85 90 95 Gly Asp Leu PheAsp Lys Ile Glu Pro Asp Val Gly Ile Asp Glu Asp 100 105 110 Val Ala GlnPhe Tyr Phe Ala Gln Leu Met Glu Gly Ile Ser Phe Met 115 120 125 His SerLys Gly Val Ala His Arg Asp Leu Lys Pro Glu Asn Ile Leu 130 135 140 LeuAsp Tyr Asn Gly Asn Leu Lys Ile Ser Asp Phe Gly Phe Ala Ser 145 150 155160 Leu Phe Ser Tyr Lys Gly Lys Ser Arg Leu Leu Asn Ser Pro Val Gly 165170 175 Ser Pro Pro Tyr Ala Ala Pro Glu Ile Thr Gln Gln Tyr Asp Gly Ser180 185 190 Lys Val Asp Val Trp Ser Cys Gly Ile Ile Leu Phe Ala Leu LeuLeu 195 200 205 Gly Asn Thr Pro Trp Asp Glu Ala Ile Ser Asn Thr Gly AspTyr Leu 210 215 220 Leu Tyr Lys Lys Gln Cys Glu Arg Pro Ser Tyr His ProTrp Asn Leu 225 230 235 240 Leu Ser Pro Gly Ala Tyr Ser Ile Ile Thr GlyMet Leu Arg Ser Asp 245 250 255 Pro Phe Lys Arg Tyr Ser Val Lys His ValVal Gln His Pro Trp Leu 260 265 270 Thr Ser Ser Thr Pro Phe Arg Thr LysAsn Gly Asn Cys Ala Asp Pro 275 280 285 Val Ala Leu Ala Ser Arg Leu MetLeu Lys Leu Arg Ile Asp Leu Asp 290 295 300 Lys Pro Arg Leu Ala Ser SerArg Ala Ser Gln Asn Asp Ser Gly Phe 305 310 315 320 Ser Met Thr Gln ProAla Phe Lys Lys Asn Asp Gln Lys Glu Leu Asp 325 330 335 Arg Val Glu ValTyr Gly Ala Leu Ser Gln Pro Val Gln Leu Asn Lys 340 345 350 Asn Ile AspVal Thr Glu Ile Leu Glu Lys Asp Pro Ser Leu Ser Gln 355 360 365 Phe CysGlu Asn Glu Gly Phe Ile Lys Arg Leu Ala Lys Lys Ala Lys 370 375 380 AsnPhe Tyr Glu Ile Cys Pro Pro Glu Arg Leu Thr Arg Phe Tyr Ser 385 390 395400 Arg Ala Ser Arg Glu Thr Ile Ile Asp His Leu Tyr Asp Ser Leu Arg 405410 415 Leu Leu Ala Ile Ser Val Thr Met Lys Tyr Val Arg Asn Gln Thr Ile420 425 430 Leu Tyr Val Asn Leu His Asp Lys Arg Lys Cys Leu Leu Gln GlyVal 435 440 445 Ile Glu Leu Thr Asn Leu Gly His Asn Leu Glu Leu Ile AsnPhe Ile 450 455 460 Lys Arg Asn Gly Asp Pro Leu Glu Trp Arg Lys Phe PheLys Asn Val 465 470 475 480 Val Ser Ser Ile Gly Lys Pro Ile Val Leu ThrAsp Val Ser Gln Asn 485 490 495 <210> SEQ ID NO 4 <211> LENGTH: 235<212> TYPE: DNA <213> ORGANISM: HOMO SAPIENS <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: 44, 51, 63, 82, 129, 153, 161, 197,199, 200, 217, 224, 231 <223> OTHER INFORMATION: a or g or c or t,unknown, or other <220> FEATURE: <223> OTHER INFORMATION: 046081H1,CORNNOT01 <400> SEQUENCE: 4 gcaaaggaca gtccgccgag gtgctcggtg gagtcatggcattncccttt ntggaagact 60 ggnccttggt gcaaaccctg gnagaaggtg cctatggagaagttcaactt gctgtaaata 120 gagtaactna agaagcagtc gcagtgaaga ttntagatatnaagcgtgcc gtagactgtc 180 ccgaaaatat taagtangnn atctgtatca ataaaantgctaantcatga naatt 235 <210> SEQ ID NO 5 <211> LENGTH: 342 <212> TYPE: DNA<213> ORGANISM: HOMO SAPIENS <220> FEATURE: <221> NAME/KEY: unsure <222>LOCATION: 26, 48, 59, 69, 98, 166, 169, 200, 221, 224, 227, 231, 240,<220> FEATURE: <222> LOCATION: 241,242, 247, 251, 252, 257, 260, 267,284, 292, 296, 303, <220> FEATURE: <222> LOCATION: 305, 306, 319, 337,339 <223> OTHER INFORMATION: a or g or c or t, unknown, or other <223>OTHER INFORMATION: 046081F1, CORNNOT01 <400> SEQUENCE: 5 gccccaaagttgatacaatt ttattngaaa accaactttt tgtaagtnta acaggtaang 60 gaccaaggntcagtctattt tcttatagtt ccttactntt ccttcctctt tattcttata 120 cctaaagtttgtcaccagta gttgaaaggt aaatatggtt tgaagnggna atttgggaaa 180 tttttagggtgttcaaaggn atttttgggg aaagttggta ngcnctnggg ntttgcaacn 240 nntaagncaangntttnctn cctccantgg gaaacacctt cttnaatttt anttancatt 300 ttntnnaaaccgggttttnt tcctggcccg gcccttngng tt 342 <210> SEQ ID NO 6 <211> LENGTH:863 <212> TYPE: DNA <213> ORGANISM: HOMO SAPIENS <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: 3, 13, 17, 21, 27, 31, 85, 89, 103,117, 142, 165, 167, 171, 175, <220> FEATURE: <222> LOCATION: 229, 266,274, 287, 288, 307, 326, 403, 502, 533, 544, 546, 551, <220> FEATURE:<222> LOCATION: 562, 563, 569, 594, 605, 643, 652, 655, 671, 688, 689,706, 708, <220> FEATURE: <222> LOCATION: 711, 719, 721, 731, 736, 741,749, 751, 755, 756, 758, 760, 772, <220> FEATURE: <222> LOCATION: 775,779, 780, 786, 787, 788, 794, 796, 804, 808, 813, 825, 829, <220>FEATURE: <222> LOCATION: 830, 836, 838, 841, 849, 858, 860 <223> OTHERINFORMATION: a or g or c or t, unknown, or other <223> OTHERINFORMATION: 046081X3, CORNNOT01 <400> SEQUENCE: 6 ctnggtcaat ctnaagncgangggccnggc nccacatgga aatgtttttt tttgaagact 60 gggacttggt gcaaacccggggagnaggnc cctaagaaaa aancagtcgc agtgaanatt 120 gtagatatga agcgtgccgtanactgtcca gaaaatatta agaantntat ntttntcaat 180 aaaatgctaa atcatgaaaatgtagtaaaa ttctatggtc acaggagana aggcaatatc 240 caatatttat ttctggagtactgtantgga gganagcttt ttgaccnncc cgagccagac 300 ataggcntgc ctgaaccaaatgctcntata ttcttccatc aactcatggc aggggtggtt 360 tatctgcatg gtattggaataactcacagg gatattaaac canaaaaatc ttctgttgga 420 tgaaagggat aacctcaaaatctcagactt tggcttggca acagtatttc ggtataataa 480 tcgtgagcgt ttgttgaacaanatgtgtgg tactttacca tatgttgctc canaacttct 540 gaananaaaa naattccatgcnnaaccant ttgatgtttg gtcctgtgga atantactta 600 ctgcnatgct cgctggagaattgccatggg accaacccac ganagctgtc angantattc 660 tgactggaaa naaaaaaaaacatactcnnc ccttggaaaa aaatcnantc ngctcctcna 720 nctctgccgc ntaaancctanttgaaatnc ntccnncnan aataccttcc cnaantccnn 780 aaaaannntg gtcncnccccccanaaangg ggnaaaggcc caatncctnn ggtgtntntc 840 naatccccnt ggattccnancac 863 <210> SEQ ID NO 7 <211> LENGTH: 218 <212> TYPE: DNA <213>ORGANISM: HOMO SAPIENS <220> FEATURE: <221> NAME/KEY: unsure <222>LOCATION: 27, 50, 172 <223> OTHER INFORMATION: a or g or c or t,unknown, or other <220> FEATURE: <223> OTHER INFORMATION: 516219H1,MMLR1DT01 <400> SEQUENCE: 7 cttctgttgg atgaaaggga taacctnaaa atctcagactttggcttggn aacagtattt 60 cggtataata atcgtgagcg tttgttgaac aagatgtgtggtactttacc atatgttgct 120 ccagaacttc tgaagagaag agatttcatg cagaaccagttgatgtttgg tnctgtggaa 180 tagtacttac tgcaatgctc gctggagaat tgccatgg 218<210> SEQ ID NO 8 <211> LENGTH: 323 <212> TYPE: DNA <213> ORGANISM: HOMOSAPIENS <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 255, 263,319, 321 <223> OTHER INFORMATION: a or g or c or t, unknown, or other<220> FEATURE: <223> OTHER INFORMATION: 516219F1, MMLR1DT01 <400>SEQUENCE: 8 aggattcccc agagccgatg gtccgatcat gtggcaggaa gccaaaccttctggctgctc 60 acatatcaat cagcttccct ttaatcttca ggaagtgtct cttgaactccaatccatcac 120 ccttagaaag ccggaagtca accaatattt tatcatccat ttctaacaaattcactttga 180 aaatgagttt attgtttctc ctatcagttg ttgatatagt aacctgattcatacaacttt 240 tcttccattg atagnccaac ttntcacaag tctctttcag gcattgataggtttgtctgc 300 atccatttgg taaaggatng ngt 323 <210> SEQ ID NO 9 <211>LENGTH: 519 <212> TYPE: DNA <213> ORGANISM: HOMO SAPIENS <220> FEATURE:<221> NAME/KEY: unsure <222> LOCATION: 3, 11, 25, 216, 242, 363, 364,399, 431, 499, 515, 516 <223> OTHER INFORMATION: a or g or c or t,unknown, or other <220> FEATURE: <223> OTHER INFORMATION: 516219X5,MMLR1DT01 <400> SEQUENCE: 9 tanaggcaaa ngacagtccc cccangtgct tggtggagtcatggcagtgc cctttgtgga 60 agactgggac ttggtgcaaa ccctgggaga aggtgcctatggagaagttc aacttgctgt 120 gaatagagta actgaagaag cagtcgcagt gaagattgtagatatgaagc gtgccgtaga 180 ctgtccagaa aatattaaga aagagatctg tatcantaaaatgctaaatc atgaaaatgt 240 antaaaattc tatggtcaca ggagagaagg caatatccaatatttatttc tggagtactg 300 tagtggagga gagctttttg acagaataga gccagacataggcatgcctg aaccagatgc 360 tcnnagattc ttccatcaac tcatggcagg ggtggtttntctgcatggta ttggaataac 420 tcacggggat nttaaaccag aaaatcttct gttggatgaaagggataacc tcaaaatctc 480 agactttggc ttggcaacng tatttcggta taatnnggg 519<210> SEQ ID NO 10 <211> LENGTH: 357 <212> TYPE: DNA <213> ORGANISM:HOMO SAPIENS <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 46<223> OTHER INFORMATION: a or g or c or t, unknown, or other <220>FEATURE: <223> OTHER INFORMATION: 2044650H1, THP1T7T01 <400> SEQUENCE:10 ggggtttaat atccctgtga gttattccaa taccatgcag ataaancacc cctgccatga 60gttgatggaa gaatctctga gcatctggtt caggcatgcc tatgtctggc tctattctgt 120caaaaagctc tcctccacta cagtactcca gaaataaata ttggatattg ccttctctcc 180tgtgaccata gaattttact acattttcat gatttagcat tttattgata cagatctctt 240tcttaatatt ttctggacag tctacggcac gcttcatatc tacaatcttc actgcgactg 300ttcttcagtt actctattca cagcaagttg aattctccca taggcacctc gagccgg 357 <210>SEQ ID NO 11 <211> LENGTH: 286 <212> TYPE: DNA <213> ORGANISM: HOMOSAPIENS <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 45, 49,58, 59, 62, 63, 66, 68 <223> OTHER INFORMATION: a or g or c or t,unknown, or other <220> FEATURE: <223> OTHER INFORMATION: 2731758H1,OVARTUT04 <400> SEQUENCE: 11 tgcatttgga ttcctgcagt ggtgggcaaa ggacagtccgccgangtgnt cggtggannc 60 cnnggnantg ccctttgtgg aagactggga cttggtgcaaaccctgggag aaggtgccta 120 tggagaagtt caacttgctg tgaatagagt aactgaagaagcagtcgcag tgaagattgt 180 agatatgaag cgtgccgtag actgtccaga aaatattaagaaagagatct gtatcaataa 240 aatgctaaat catgaaaatg tagtaaaatt ctatggtcacaggaga 286 <210> SEQ ID NO 12 <211> LENGTH: 22 <212> TYPE: PRT <213>ORGANISM: HOMO SAPIENS <400> SEQUENCE: 12 Cys Gly Lys Ile Lys Gly LysLeu Ile Asp Ile Val Ser Ser Gln Lys 1 5 10 15 Val Trp Leu Pro Ala Thr 20<210> SEQ ID NO 13 <211> LENGTH: 87 <212> TYPE: DNA <213> ORGANISM: HOMOSAPIENS <400> SEQUENCE: 13 gtgaattcac caccatggca gtgccctttg tggaagactgggacttggtg caaaccctgg 60 gagaaggtgc ctatggagaa gttcaac 87 <210> SEQ IDNO 14 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: HOMO SAPIENS<400> SEQUENCE: 14 cactcgagga ttccccagag ccgatggtc 29 <210> SEQ ID NO 15<211> LENGTH: 57 <212> TYPE: DNA <213> ORGANISM: HOMO SAPIENS <400>SEQUENCE: 15 ggaataactc acagggatat taaaccagaa aatcttctgt tggatgaaagggataac 57 <210> SEQ ID NO 16 <211> LENGTH: 1479 <212> TYPE: DNA <213>ORGANISM: HOMO SAPIENS <400> SEQUENCE: 16 gaattcggct tccaccatggcagtgccctt tgtggaagac tgggacttgg tgcaaaccct 60 gggagaaggt gcctatggagaagttcaact tgctgtgaat agagtaactg aagaagcagt 120 cgcagtggcg attgtagatatgaagcgtgc cgtagactgt ccagaaaata ttaagaaaga 180 gatctgtatc aataaaatgctaaatcatga aaatgtagta aaattctatg gtcacaggag 240 agaaggcaat atccaatatttatttctgga gtactgtagt ggaggagagc tttttgacag 300 aatagagcca gacataggcatgcctgaacc agatgctcag agattcttcc atcaactcat 360 ggcaggggtg gtttatctgcatggtattgg aataactcac agggatatta aaccagaaaa 420 tcttctgttg gatgaaagggataacctcaa aatctcagac tttggcttgg caacagtatt 480 tcggtataat aatcgtgagcgtttgttgaa caagatgtgt ggtactttac catatgttgc 540 tccagaactt ctgaagagaagagaatttca tgcagaacca gttgatgttt ggtcctgtgg 600 aatagtactt actgcaatgctcgctggaga attgccatgg gaccaaccca gtgacagctg 660 tcaggagtat tctgactggaaagaaaaaaa aacatacctc aacccttgga aaaaaatcga 720 ttctgctcct ctagctctgctgcataaaat cttagttgag aatccatcag caagaattac 780 cattccagac atcaaaaaagatagatggta caacaaaccc ctcaagaaag gggcaaaaag 840 gccccgagtc acttcaggtggtgtgtcaga gtctcccagt ggattttcta agcacattca 900 atccaatttg gacttctctccagtaaacag tgcttctagt gaagaaaatg tgaagtactc 960 cagttctcag ccagaaccccgcacaggtct ttccttatgg gataccagcc cctcatacat 1020 tgataaattg gtacaagggatcagcttttc ccagcccaca tgtcctgatc atatgctttt 1080 gaatagtcag ttacttggcaccccaggatc ctcacagaac ccctggcagc ggttggtcaa 1140 aagaatgaca cgattctttaccaaattgga tgcagacaaa tcttatcaat gcctgaaaga 1200 gacttgtgag aagttgggctatcaatggaa gaaaagttgt atgaatcagg ttactatatc 1260 aacaactgat aggagaaacaataaactcat tttcaaagtg aatttgttag aaatggatga 1320 taaaatattg gttgacttccggctttctaa gggtgatgga ttggagttca agagacactt 1380 cctgaagatt aaagggaagctgattgatat tgtgagcagc cagaaggttt ggcttcctgc 1440 cacatgatcg gaccatcggctctggggaat cctcgagtg 1479

What is claimed is:
 1. An isolated polypeptide comprising an amino acidsequence selected from the group consisting of: a) a polypeptidecomprising an amino acid sequence of SEQ ID NO:1, b) a naturallyoccurring polypeptide comprising an amino acid sequence at least 90%identical to an amino acid sequence of SEQ ID NO:1, c) a biologicallyactive fragment of a polypeptide having an amino acid sequence of SEQ IDNO:1, and d) an immunogenic fragment of a polypeptide having an aminoacid sequence of SEQ ID NO:1.
 2. An isolated polypeptide of claim 1,having a sequence of SEQ ID NO:1.
 3. An isolated polynucleotide encodinga polypeptide of claim
 1. 4. An isolated polynucleotide encoding apolypeptide of claim
 2. 5. An isolated polynucleotide of claim 4, havinga sequence of SEQ ID NO:2.
 6. A recombinant polynucleotide comprising apromoter sequence operably linked to a polynucleotide of claim
 3. 7. Acell transformed with a recombinant polynucleotide of claim
 6. 8. Atransgenic organism comprising a recombinant polynucleotide of claim 6.9. A method for producing a polypeptide of claim 1, the methodcomprising: a) culturing a cell under conditions suitable for expressionof the polypeptide, wherein said cell is transformed with a recombinantpolynucleotide, and said recombinant polynucleotide comprises a promotersequence operably linked to a polynucleotide encoding the polypeptide ofclaim 1, and b) recovering the polypeptide so expressed.
 10. A method ofclaim 9, wherein the polypeptide has the sequence of SEQ ID NO:1.
 11. Anisolated antibody which specifically binds to a polypeptide of claim 1.12. An isolated polynucleotide comprising a sequence selected from thegroup consisting of: a) a polynucleotide comprising a polynucleotidesequence of SEQ ID NO:2, b) a naturally occurring polynucleotidecomprising a polynucleotide sequence at least 90% identical to apolynucleotide sequence of SEQ ID NO:2, c) a polynucleotide having asequence complementary to a polynucleotide of a), d) a polynucleotidehaving a sequence complementary to a polynucleotide of b) and e) an RNAequivalent of a)-d).
 13. An isolated polynucleotide comprising at least60 contiguous nucleotides of a polynucleotide of claim
 12. 14. A methodfor detecting a target polynucleotide in a sample, said targetpolynucleotide having a sequence of a polynucleotide of claim 12, themethod comprising: a) hybridizing the sample with a probe comprising atleast 20 contiguous nucleotides comprising a sequence complementary tosaid target polynucleotide in the sample, and which probe specificallyhybridizes to said target polynucleotide, under conditions whereby ahybridization complex is formed between said probe and said targetpolynucleotide or fragments thereof, and b) detecting the presence orabsence of said hybridization complex, and, optionally, if present, theamount thereof.
 15. A method of claim 14, wherein the probe comprises atleast 60 contiguous nucleotides.
 16. A method for detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 12, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 17. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 18. Acomposition of claim 17, wherein the polypeptide has an amino acidsequence of SEQ ID NO:1.
 19. A method for treating a disease orcondition associated with decreased expression of functional HChk1,comprising administering to a patient in need of such treatment thecomposition of claim
 17. 20. A method for screening a compound foreffectiveness as an agonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting agonist activity in the sample.
 21. Acomposition comprising an agonist compound identified by a method ofclaim 20 and a pharmaceutically acceptable excipient.
 22. A method fortreating a disease or condition associated with decreased expression offunctional HChk1, comprising administering to a patient in need of suchtreatment a composition of claim
 21. 23. A method for screening acompound for effectiveness as an antagonist of a polypeptide of claim 1,the method comprising: a) exposing a sample comprising a polypeptide ofclaim 1 to a compound, and b) detecting antagonist activity in thesample.
 24. A composition comprising an antagonist compound identifiedby a method of claim 23 and a pharmaceutically acceptable excipient. 25.A method for treating a disease or condition associated withoverexpression of functional HChk1, comprising administering to apatient in need of such treatment a composition of claim
 24. 26. Amethod of screening for a compound that specifically binds to thepolypeptide of claim 1, the method comprising: a) combining thepolypeptide of claim 1 with at least one test compound under suitableconditions, and b) detecting binding of the polypeptide of claim 1 tothe test compound, thereby identifying a compound that specificallybinds to the polypeptide of claim
 1. 27. A method of screening for acompound that modulates the activity of the polypeptide of claim 1, saidmethod comprising: a) combining the polypeptide of claim 1 with at leastone test compound under conditions permissive for the activity of thepolypeptide of claim 1, b) assessing the activity of the polypeptide ofclaim 1 in the presence of the test compound, and c) comparing theactivity of the polypeptide of claim 1 in the presence of the testcompound with the activity of the polypeptide of claim 1 in the absenceof the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 28. A method for screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a polynucleotide sequence of claim 5, themethod comprising: a) exposing a sample comprising the targetpolynucleotide to a compound, under conditions suitable for theexpression of the target polynucleotide, b) detecting altered expressionof the target polynucleotide, and c) comparing the expression of thetarget polynucleotide in the presence of varying amounts of the compoundand in the absence of the compound.
 29. A method for assessing toxicityof a test compound, the method comprising: a) treating a biologicalsample containing nucleic acids with the test compound, b) hybridizingthe nucleic acids of the treated biological sample with a probecomprising at least 20 contiguous nucleotides of a polynucleotide ofclaim 12 under conditions whereby a specific hybridization complex isformed between said probe and a target polynucleotide in the biologicalsample, said target polynucleotide comprising a polynucleotide sequenceof a polynucleotide of claim 12 or fragment thereof, c) quantifying theamount of hybridization complex, and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 30. Adiagnostic test for a condition or disease associated with theexpression of HChk1 in a biological sample, the method comprising: a)combining the biological sample with an antibody of claim 11, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex, and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 31. The antibody of claim 11, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 32. Acomposition comprising an antibody of claim 11 and an acceptableexcipient.
 33. A method of diagnosing a condition or disease associatedwith the expression of HChk1 in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 32. 34. Acomposition of claim 32, wherein the antibody is labeled.
 35. A methodof diagnosing a condition or disease associated with the expression ofHChk1 in a subject, comprising administering to said subject aneffective amount of the composition of claim
 34. 36. A method ofpreparing a polyclonal antibody with the specificity of the antibody ofclaim 11, the method comprising: a) immunizing an animal with apolypeptide having an amino acid sequence of SEQ ID NO:1, or animmunogenic fragment thereof, under conditions to elicit an antibodyresponse, b) isolating antibodies from said animal, and c) screening theisolated antibodies with the polypeptide, thereby identifying apolyclonal antibody which binds specifically to a polypeptide having anamino acid sequence of SEQ ID NO:1.
 37. An antibody produced by a methodof claim
 36. 38. A composition comprising the antibody of claim 37 and asuitable carrier.
 39. A method of making a monoclonal antibody with thespecificity of the antibody of claim 11, the method comprising: a)immunizing an animal with a polypeptide having an amino acid sequence ofSEQ ID NO:1, or an immunogenic fragment thereof, under conditions toelicit an antibody response, b) isolating antibody producing cells fromthe animal, c) fusing the antibody producing cells with immortalizedcells to form monoclonal antibody-producing hybridoma cells, d)culturing the hybridoma cells, and e) isolating from the culturemonoclonal antibody which binds specifically to a polypeptide having anamino acid sequence of SEQ ID NO:1.
 40. A monoclonal antibody producedby a method of claim
 39. 41. A composition comprising the antibody ofclaim 40 and a suitable carrier.
 42. The antibody of claim 11, whereinthe antibody is produced by screening a Fab expression library.
 43. Theantibody of claim 11, wherein the antibody is produced by screening arecombinant immunoglobulin library.
 44. A method of detecting apolypeptide having an amino acid sequence of SEQ ID NO:1 in a sample,the method comprising: a) incubating the antibody of claim 11 with asample under conditions to allow specific binding of the antibody andthe polypeptide, and b) detecting specific binding, wherein specificbinding indicates the presence of a polypeptide having an amino acidsequence of SEQ ID NO:1 in the sample.
 45. A method of purifying apolypeptide having an amino acid sequence of SEQ ID NO:1 from a sample,the method comprising: a) incubating the antibody of claim 11 with asample under conditions to allow specific binding of the antibody andthe polypeptide, and b) separating the antibody from the sample andobtaining the purified polypeptide having an amino acid sequence of SEQID NO:1.
 46. A microarray wherein at least one element of the microarrayis a polynucleotide of claim
 13. 47. A method of generating anexpression profile of a sample which contains polynucleotides, themethod comprising: a) labeling the polynucleotides of the sample, b)contacting the elements of the microarray of claim 46 with the labeledpolynucleotides of the sample under conditions suitable for theformation of a hybridization complex, and c) quantifying the expressionof the polynucleotides in the sample.
 48. An array comprising differentnucleotide molecules affixed in distinct physical locations on a solidsubstrate, wherein at least one of said nucleotide molecules comprises afirst oligonucleotide or polynucleotide sequence specificallyhybridizable with at least 30 contiguous nucleotides of a targetpolynucleotide, and wherein said target polynucleotide is apolynucleotide of claim
 12. 49. An array of claim 48, wherein said firstoligonucleotide or polynucleotide sequence is completely complementaryto at least 30 contiguous nucleotides of said target polynucleotide. 50.An array of claim 48, wherein said first oligonucleotide orpolynucleotide sequence is completely complementary to at least 60contiguous nucleotides of said target polynucleotide.
 51. An array ofclaim 48, wherein said first oligonucleotide or polynucleotide sequenceis completely complementary to said target polynucleotide.
 52. An arrayof claim 48, which is a microarray.
 53. An array of claim 48, furthercomprising said target polynucleotide hybridized to a nucleotidemolecule comprising said first oligonucleotide or polynucleotidesequence.
 54. An array of claim 48, wherein a linker joins at least oneof said nucleotide molecules to said solid substrate.
 55. An array ofclaim 48, wherein each distinct physical location on the substratecontains multiple nucleotide molecules, and the multiple nucleotidemolecules at any single distinct physical location have the samesequence, and each distinct physical location on the substrate containsnucleotide molecules having a sequence which differs from the sequenceof nucleotide molecules at another distinct physical location on thesubstrate.
 56. A polynucleotide of claim 12, comprising thepolynucleotide sequence of SEQ ID NO:2.
 57. A polynucleotide of claim12, comprising the polynucleotide sequence of SEQ ID NO:16.